Effectiveness of Cathelicidin Antimicrobial Peptide against Ictalurid Catfish Bacterial Pathogens.

One of the major goals in aquaculture is to protect fish against infectious diseases as disease outbreaks could lead to economic losses if not controlled. Antimicrobial peptides (AMPs), a class of highly conserved peptides known to possess direct antimicrobial activities against invading pathogens, were evaluated for their ability to protect Channel Catfish Ictalurus punctatus and hybrid catfish (female Channel Catfish × male Blue Catfish I. furcatus) against infection caused by the fish pathogen Aeromonas hydrophila ML09-119. To identify effective peptides, the minimum inhibitory concentrations against bacterial pathogens Edwardsiella ictaluri S97-773, Edwardsiella piscicida E22-10, A. hydrophila ML09-119, Aeromonas veronii 03X03876, and Flavobacterium columnare GL-001 were determined in vitro. In general and overall, cathelicidins derived from alligator and sea snake exhibited more potent and rapid antimicrobial activities against the tested catfish pathogens as compared to cecropin and pleurocidin AMPs and ampicillin, the antibiotic control. When the peptides (2.5 µg of peptide/g of fish) were injected into fish and simultaneously challenged with A. hydrophila through immersion, increased survival rates in Channel Catfish and hybrid catfish were observed in both cathelicidin (alligator and sea snake) treatments as compared to other peptides and the infected control (P < 0.001) with alligator cathelicidin being the overall best treatment. Bacterial numbers in the kidney and liver of Channel Catfish and hybrid catfish also decreased (P < 0.05) for cathelicidin-injected groups at 24 and 48 h after challenge infection. These results show the potential of cathelicidin to protect catfish against bacterial infections and suggest that an approach overexpressing the peptide in transgenic fish, which is the long-term goal of this research program, may provide a method of decreasing bacterial disease problems in catfish as delivering the peptides via individual injection or feeding would not be economically feasible.

CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome.

CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp ß-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P1 individuals harboring the transgene, and high transgene expression was observed in the blood of some P1 fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish.

Cathelicidins enhance protection of channel catfish, Ictalurus punctatus, and channel catfish ♀ × blue catfish, Ictalurus furcatus ♂ hybrid catfish against Edwarsiella ictaluri infection.

Cathelicidins are a class of antimicrobial peptides (AMPs) known to possess rapid and direct antimicrobial activities against a variety of microorganisms. Recently identified cathelicidins derived from alligator and sea snake were found to be more effective in inhibiting microbial growth than other AMPs previously characterized. The ability of these two cathelicidins along with the peptides, cecropin and pleurocidin, to protect channel catfish (Ictalurus punctatus, Rafinesque) and hybrid catfish (I. punctatus ♀ × blue catfish, Ictalurus furcatus, Valenciennes ♂) against Edwardsiella ictaluri, one of the most prevalent pathogens affecting commercial catfish industry, was investigated. Cathelicidin-injected fish (50 µg ml-1  fish-1 ) that were simultaneously challenged with E. ictaluri through bath immersion at a concentration of ~1 × 106 CFU/ml had increased survival rates compared with other peptide treatments and the infected control. Bacterial numbers were also reduced in the liver and kidney of channel catfish and hybrid catfish in the cathelicidin treatments 24 hr post-infection. After 8 days of challenge, serum was collected to determine immune-related parameters such as bactericidal activity, lysozyme, serum protein, albumin and globulin. These immune-related parameters were significantly elevated in fish injected with the two cathelicidins as compared to other peptide treatments. These results indicate that cathelicidins derived from alligator and sea snake can stimulate immunity and enhance the resistance to E. ictaluri infection in channel catfish and hybrid catfish.

Classification of a Hypervirulent Aeromonas hydrophila Pathotype Responsible for Epidemic Outbreaks in Warm-Water Fishes.

Lineages of hypervirulent Aeromonas hydrophila (vAh) are the cause of persistent outbreaks of motile Aeromonas septicemia in warm-water fishes worldwide. Over the last decade, this virulent lineage of A. hydrophila has resulted in annual losses of millions of tons of farmed carp and catfish in the People's Republic of China and the United States (US). Multiple lines of evidence indicate US catfish and Asian carp isolates of A. hydrophila affiliated with sequence type 251 (ST251) share a recent common ancestor. To address the genomic context for the putative intercontinental transfer and subsequent geographic spread of this pathogen, we conducted a core genome phylogenetic analysis on 61 Aeromonas spp. genomes, of which 40 were affiliated with A. hydrophila, with 26 identified as epidemic strains. Phylogenetic analyses indicate all ST251 strains form a coherent lineage affiliated with A. hydrophila. Within this lineage, conserved genetic loci unique to A. hydrophila were identified, with some genes present in consistently higher copy numbers than in non-epidemic A. hydrophila isolates. In addition, results from analyses of representative ST251 isolates support the conclusion that multiple lineages are present within US vAh isolated from Mississippi, whereas vAh isolated from Alabama appear clonal. This is the first report of genomic heterogeneity within US vAh isolates, with some Mississippi isolates showing closer affiliation with the Asian grass carp isolate ZC1 than other vAh isolated in the US. To evaluate the biological significance of the identified heterogeneity, comparative disease challenges were conducted with representatives of different vAh genotypes. These studies revealed that isolate ZC1 yielded significantly lower mortality in channel catfish, relative to Alabama and Mississippi vAh isolates. Like other Asian vAh isolates, the ZC1 lineage contains all core genes for a complete type VI secretion system (T6SS). In contrast, more virulent US isolates retain only remnants of the T6SS (clpB, hcp, vgrG, and vasH) which may have functional implications. Collectively, these results characterize a hypervirulent A. hydrophila pathotype that affects farmed fish on multiple continents.

Dietary supplementation with Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus oryzae enhance immunity and disease resistance against Aeromonas hydrophila and Streptococcus iniae infection in juvenile tilapia Oreochromis niloticus.

A feeding trial was conducted to investigate the effects of dietary administration of probiotic with Bacillus subtilis, Aspergillus oryzae and Saccharomyces cerevisiae on growth, innate immune response, Hemato-immunological parameters and disease resistance of Nile tilapia, Oreochromis niloticus. Animals were distributed in three equal groups, each of five replicates and received one of the following experimental diets for four weeks: Control, non-supplemented diet; 5 g kg(-1) probiotic mixture (B. subtilis 1.5 × 10(9) CFU g(-1), S. cerevisiae 10(9) CFU g(-1) and A. oryzae 2 × 10(9) CFU g(-1)); and 10 g kg(-1) probiotic mixture (B. subtilis 3.0 × 10(9) CFU g(-1), S. cerevisiae 2.0 × 10(9) CFU g(-1) and A. oryzae 4.0 × 10(9) CFU g(-1)). The respiratory burst activity, white blood cells and hematological parameters were evaluated after four, five and six weeks of feeding. At the end of the growth trial, fish were sampled for intestinal microbiology and challenged by intraperitoneal injection of LD50 concentration of Aeromonas hydrophila and Streptococcus iniae. Mortality was recorded for the following 3 weeks. Results showed that administration of the probiotic had no significant effect on the growth rates of Nile tilapias, although the fish fed probiotics had better feed conversion. Respiratory burst activity, erythrocyte fragility and levels of white blood cells were significantly improved in tilapias fed diet supplemented with probiotic levels (P < 0.05), which may exhibit up-regulating effects on tilapia immune parameters. The cumulative mortality after A. hydrophila and S. iniae challenge decreased in tilapias fed with probiotic (P < 0.05). The present study demonstrated the potential of B. subtilis, S. cerevisiae and A. oryzae combined as beneficial dietary probiotic in juvenile O. niloticus.

An Asian origin of virulent Aeromonas hydrophila responsible for disease epidemics in United States-farmed catfish.

UNLABELLED: Since 2009, catfish farming in the southeastern United States has been severely impacted by a highly virulent and clonal population of Aeromonas hydrophila causing motile Aeromonas septicemia (MAS) in catfish. The possible origin of this newly emerged highly virulent A. hydrophila strain is unknown. In this study, we show using whole-genome sequencing and comparative genomics that A. hydrophila isolates from diseased grass carp in China and catfish in the United States have highly similar genomes. Our phylogenomic analyses suggest that U.S. catfish isolates emerged from A. hydrophila populations of Asian origin. Furthermore, we identified an A. hydrophila strain isolated in 2004 from a diseased catfish in Mississippi, prior to the onset of the major epidemic outbreaks in Alabama starting in 2009, with genomic characteristics that are intermediate between those of the Asian and Alabama fish isolates. Investigation of A. hydrophila strain virulence demonstrated that the isolate from the U.S. catfish epidemic is significantly more virulent to both channel catfish and grass carp than is the Chinese carp isolate. This study implicates the importation of fish or fishery products into the United States as the source of highly virulent A. hydrophila that has caused severe epidemic outbreaks in United States-farmed catfish and further demonstrates the potential for invasive animal species to disseminate bacterial pathogens worldwide. IMPORTANCE: Catfish aquaculture farming in the southeastern United States has been severely affected by the emergence of virulent Aeromonas hydrophila responsible for epidemic disease outbreaks, resulting in the death of over 10 million pounds of catfish. Because the origin of this newly emerged A. hydrophila strain is unknown, this study used a comparative genomics approach to conduct a phylogenomic analysis of A. hydrophila isolates obtained from the United States and Asia. Our results suggest that the virulent isolates from United States-farmed catfish have a recent common ancestor with A. hydrophila isolates from diseased Asian carp. We have also observed that an Asian carp isolate, like recent U.S. catfish isolates, is virulent in catfish. The results from this study suggest that the highly virulent U.S. epidemic isolates emerged from an Asian source and provide another example of the threat that invasive species pose in the dissemination of bacterial pathogens.

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.

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.

An outer membrane porin protein modulates phage susceptibility in Edwardsiella ictaluri.

Bacteriophages ΦeiAU and ΦeiDWF are lytic to the catfish pathogen Edwardsiella (Edw.) ictaluri. The Edw. ictaluri host factors that modulate phage-host interactions have not been described previously. This study identified eleven unique Edw. ictaluri host factors essential for phage infection by screening a transposon mutagenized library of two Edw. ictaluri strains for phage-resistant mutants. Two mutants were isolated with independent insertions in the ompLC gene that encodes a putative outer membrane porin. Phage binding and efficiency of plaquing assays with Edw. ictaluri EILO, its ompLC mutant and a complemented mutant demonstrated that OmpLC serves as a receptor for phage ΦeiAU and ΦeiDWF adsorption. Comparison of translated OmpLCs from 15 Edw. ictaluri strains with varying degrees of phage susceptibility revealed that amino acid variations were clustered on the predicted extracellular loop 8 of OmpLC. Deletion of loop 8 of OmpLC completely abolished phage infectivity in Edw. ictaluri. Site-directed mutagenesis and transfer of modified ompLC genes to complement the ompLC mutants demonstrated that changes in ompLC sequences affect the degree of phage susceptibility. Furthermore, Edw. ictaluri strain Alg-08-183 was observed to be resistant to ΦeiAU, but phage progeny could be produced if phage DNA was electroporated into this strain. A host-range mutant of ΦeiAU, ΦeiAU-183, was isolated that was capable of infecting strain Alg-08-183 by using OmpLC as a receptor for adsorption. The results of this study identified Edw. ictaluri host factors required for phage infection and indicated that OmpLC is a principal molecular determinant of phage susceptibility in this pathogen.

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

BACKGROUND: The bacterial pathogen Edwardsiella ictaluri 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 E. ictaluri. RESULTS: The genomes of three Edwardsiella ictaluri-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 Siphoviridae. CONCLUSIONS: These E. ictaluri-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 E. ictaluri 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.

Cloning, expression, and immunogenicity of Flavobacterium columnare 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 microg 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.

Host-specific association between Flavobacterium columnare genomovars and fish species.

A total of 90 Flavobacterium columnare isolates were recovered from predominant wild fish species in the Mobile River, Alabama, USA. Isolates were identified and confirmed by fatty acid methyl ester analysis and specific PCR amplification. Genomovar ascription was performed using 16S-restriction fragment length polymorphism (RFLP) analysis. The majority of genomovar I isolates were recovered from threadfin shad while genomovar II isolates came from catfish (including channel and blue catfish). Additional genotyping methods, including multilocus sequence analysis (MLSA), internal spacer region-single strand conformation polymorphism analysis (ISR-SSCP) and amplified fragment length polymorphism (AFLP), confirmed a clear division of the isolates into two groups that matched genomovar ascription. Fingerprinting methods revealed a higher genetic diversity within genomovar II isolates. Our data confirmed the coexistence of F. columnare genomovars I and II in a natural environment. A statistically significant association between genomovar I and threadfin shad was demonstrated while genomovar II strains were mainly recovered from catfish species.

The Epidemiology of Bacterial Diseases in Food-Size Channel Catfish.

Enteric septicemia of catfish (ESC) and columnaris are the most economically important bacterial diseases affecting the channel catfish Ictalurus punctatus industry in the United States. Although these two diseases have been extensively researched, little is known about their prevalence and epidemiology in production systems. In 1997, a two-part survey of catfish producers in Alabama, Arkansas, Louisiana, and Mississippi was conducted to estimate the proportion of ponds and catfish operations that have these diseases and to develop information on the risk factors associated with reporting an occurrence. The response rates to the two phases of the survey were 65.6% and 75.3%, respectively. Overall, 78.1% of all operations and 42.1% of all ponds experienced problems with ESC/columnaris. Higher percentages of large operations and ponds on large operations experienced these problems. The most frequently reported average loss per outbreak of the two diseases was 200-2,000 lb (1 lb = 0.454 kg) per outbreak. Univariate analysis and multivariable regression modeling of the survey data identified three possible risk factors associated with ESC/columnaris, namely, operation size, stocking density, and feeding rate. Conversely, operations that produced their own fingerlings and those that drained ponds at intervals of 3 years or less were less likely to report losses. The associations identified in this study do not establish firm causal relationships, but they do generate hypotheses about managerial and environmental interactions that represent substantial risks to production.

The Epidemiology of Bacterial Diseases in Food-Size Channel Catfish.

Enteric septicemia of catfish (ESC) and columnaris are the most economically important bacterial diseases affecting the channel catfish Ictalurus punctatus industry in the United States. Although these two diseases have been extensively researched, little is known about their prevalence and epidemiology in production systems. In 1997, a two-part survey of catfish producers in Alabama, Arkansas, Louisiana, and Mississippi was conducted to estimate the proportion of ponds and catfish operations that have these diseases and to develop information on the risk factors associated with reporting an occurrence. The response rates to the two phases of the survey were 65.6% and 75.3%, respectively. Overall, 78.1% of all operations and 42.1% of all ponds experienced problems with ESC/columnaris. Higher percentages of large operations and ponds on large operations experienced these problems. The most frequently reported average loss per outbreak of the two diseases was 200-2,000 lb (1 lb = 0.454 kg) per outbreak. Univariate analysis and multivariable regression modeling of the survey data identified three possible risk factors associated with ESC/columnaris, namely, operation size, stocking density, and feeding rate. Conversely, operations that produced their own fingerlings and those that drained ponds at intervals of 3 years or less were less likely to report losses. The associations identified in this study do not establish firm causal relationships, but they do generate hypotheses about managerial and environmental interactions that represent substantial risks to production.