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.

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.

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.

Early mucosal responses in blue catfish (Ictalurus furcatus) skin to Aeromonas hydrophila infection.

Bacterial pathogens are well-equipped to detect, adhere to, and initiate infection in their finfish hosts. The mucosal surfaces of fish, such as the skin, function as the front line of defense against such bacterial insults that are routinely encountered in the aquatic environment. While recent progress has been made, and despite the obvious importance of mucosal surfaces, the precise molecular events that occur soon after encountering bacterial pathogens remain unclear. Indeed, these early events are critical in mounting appropriate responses that ultimately determine host survival or death. In the present study, we investigated the transcriptional consequences of a virulent Aeromonas hydrophila challenge in the skin of blue catfish, Ictalurus furcatus. We utilized an 8×60K Agilent microarray to examine gene expression profiles at key early timepoints following challenge (2 h, 12 h, and 24 h). A total of 1155 unique genes were significantly altered during at least one timepoint. We observed dysregulation in a number of genes involved in diverse pathways including those involved in antioxidant responses, apoptosis, cytoskeletal rearrangement, immunity, and extracellular matrix protein diversity and regulation. Taken together, A. hydrophila coordinately modulates mucosal factors across numerous cellular pathways in a manner predicted to enhance its ability to adhere to and infect the blue catfish host.

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.

RNA-seq analysis of mucosal immune responses reveals signatures of intestinal barrier disruption and pathogen entry following Edwardsiella ictaluri infection in channel catfish, Ictalurus punctatus.

The mucosal surfaces of fish (gill, skin, gastrointestinal tract) are important sites of bacterial exposure and host defense mechanisms. In mammalian systems, the intestinal epithelium is well characterized as both a selectively permeable barrier regulated by junctional proteins and as a primary site of infection for a number of enteric pathogens including viruses, bacteria, and parasites. The causative bacterium of enteric septicemia of catfish, Edwardsiella ictaluri, is believed to gain entry through the intestinal epithelium, with previous research using a rat intestinal epithelial cell line (IEC-6) indicating actin polymerization and receptor-mediated endocytosis as potential mechanisms of uptake. Here, we utilized high-throughput RNA-seq to characterize the role of the intestinal epithelial barrier following E. ictaluri challenge. A total of 197.6 million reads were obtained and assembled into 176,481 contigs with an average length of 893.7 bp and N50 of 1676 bp. The assembled contigs contained 14,457 known unigenes, including 2719 genes not previously identified in other catfish transcriptome studies. Comparison of digital gene expression between challenged and control samples revealed 1633 differentially expressed genes at 3 h, 24 h, and 3 day following exposure. Gene pathway analysis of the differentially expressed gene set indicated the centrality of actin cytoskeletal polymerization/remodelling and junctional regulation in pathogen entry and subsequent inflammatory responses. The expression patterns of fifteen differentially expressed genes related to intestinal epithelial barrier dysfunction were validated by quantitative real-time RT-PCR (average correlation coeff. 0.92, p < 0.001). Our results set a foundation for future studies comparing mechanisms of pathogen entry and mucosal immunity across several important catfish pathogens including E. ictaluri, Edwardsiellatarda, Flavobacterium columnare, and virulent atypical Aeromonas hydrophila. Understanding of molecular mechanisms of pathogen entry during infection will provide insight into strategies for selection of resistant catfish brood stocks against various diseases.

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.

Molecular responses of ceruloplasmin to Edwardsiella ictaluri infection and iron overload in channel catfish (Ictalurus punctatus).

Ceruloplasmin is a serum ferroxidase that carries more than 90% of the copper in plasma and has documented roles in iron homeostasis as well as antioxidative functions. In our previous studies, it has been shown that the ceruloplasmin gene is strongly up-regulated in catfish during challenge with Edwardsiella ictaluri. However, little is known about the function of this gene in teleost fish. The objective of this study, therefore, was to characterize the ceruloplasmin gene from channel catfish, determine its genomic organization, profile its patterns of tissue expression, and establish its potential for physiological antioxidant responses in catfish after bacterial infection with E. ictaluri and iron treatment. The genomic organization suggested that the catfish ceruloplasmin gene had 20 exons and 19 introns, encoding 1074 amino acids. Exon sizes of the catfish ceruloplasmin gene were close to or identical with mammalian and zebrafish homologs. Further phylogenetic analyses suggested that the gene was highly conserved through evolution. The catfish ceruloplasmin gene was mapped to both the catfish physical map and linkage map. The catfish ceruloplasmin gene was mainly expressed in liver with limited expression in other tissues, and it was significantly up-regulated in the liver after bacterial infection alone or after co-injection with bacteria and iron-dextran, while expression was not significantly induced with iron-dextran treatment alone.

Structure and expression of transferrin gene of channel catfish, Ictalurus punctatus.

Transferrin is important in iron metabolism and has been reported to be involved in disease defence responses after bacterial infection. In this study, we identified, sequenced, and characterized the transferrin gene from channel catfish, Ictalurus punctatus. The catfish transferrin gene was similar to those of other vertebrate species with 17 exons and 16 introns. Sequence analysis indicated the presence of the two duplicated lobes, each containing two sub-domains separated by a cleft harboring the iron-binding site, suggesting their structural conservation. The channel catfish transferrin cDNA encodes 679 amino acids with 42-56% similarity to known transferrin genes from various species. Southern blot analysis suggested the presence of two copies of the transferrin gene in the catfish genome, perhaps arranged in a tandem fashion. The catfish transferrin gene was mapped to a catfish BAC-based physical map. The catfish transferrin gene was highly expressed in the liver, but expression was low in most other tested tissues. Transferrin expression was significantly up-regulated after infection with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish. Such induction was also found with co-injection of iron-dextran and E. ictaluri, while transferrin expression was not significantly induced with the injection of iron-dextran alone.

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.

The warm temperature acclimation protein Wap65 as an immune response gene: its duplicates are differentially regulated by temperature and bacterial infections.

The warm temperature acclimation related 65kDa protein (Wap65) in teleost fish shares high structural similarities with mammalian hemopexins. Recent studies using microarray analysis indicated that this temperature acclimation protein may also be involved in immune responses. To provide evidence of its potential involvement in immune responses after bacterial infections, we have identified and characterized two types of Wap65 genes in channel catfish, referred to as Wap65-1 and Wap65-2, respectively. While Wap65-1 and Wap65-2 are both structurally similar to the mammalian hemopexins, they exhibit highly differential patterns of spatial expression. Wap65-1 was expressed in a wide range of tissues, whereas Wap65-2 was only expressed in the liver. Their regulation with warm temperature and bacterial infections was also highly different: Wap65-1 was constitutively expressed, whereas Wap65-2 was highly regulated by both warm temperature and bacterial infections, and warm temperature and bacterial infections appeared to synergistically induce the expression of Wap65-2. The great contrast of expression patterns and regulation of the two catfish Wap65 genes suggested both neofunctionalization and partitioning of their functions. Phylogenetic analysis indicated that the duplicated catfish Wap65 genes were evolved not only from whole genome duplication, but also from tandem, intrachromosomal gene duplications. Taken together, the results of this study suggest that Wap65 genes are not only important for its classical role as a warm temperature acclimation protein, but more importantly, may also function as an immune response protein.

Microarray analysis of gene expression in the blue catfish liver reveals early activation of the MHC class I pathway after infection with Edwardsiella ictaluri.

The acute nature of disease outbreaks in aquaculture settings has served to emphasize the importance of the innate immune response of fish for survival and led to the recent identification and characterization of many of its components. Catfish, the predominant aquaculture species in the United States, is an important model for the study of the teleost immune system. However, transcriptomic-level studies of disease-related gene expression in catfish have only recently been initiated, and understanding of immune responses to pathogen infections is limited. Here, we have developed and utilized a 28K in situ oligonucleotide microarray composed of blue catfish (Ictalurus furcatus) and channel catfish (Ictalurus punctatus) transcripts. While channel catfish accounts for the majority of commercial production, the closely related blue catfish possesses several economically important phenotypic traits. Microarray analysis of gene expression changes in blue catfish liver after infection with Gram-negative bacterium Edwardsiella ictaluri indicated the strong upregulation of several pathways involved in the inflammatory immune response and potentially in innate disease resistance. A multifaceted response to infection could be observed, encompassing the complement cascade, iron regulation, inflammatory cell signaling, and antigen processing and presentation. The induction of several components of the MHC class I-related pathway following infection with an intracellular bacterium is reported here for the first time in fish. A comparison with previously published expression profiles in the channel catfish liver was also made and the microarray results extended by use of quantitative RT-PCR. Our results add to the understanding of the teleost immune responses and provide a solid foundation for future functional characterization, genetic mapping, and QTL analysis of immunity-related genes from catfish.

Impacts of florfenicol on the microbiota landscape and resistome as revealed by metagenomic analysis.

BACKGROUND: Drug-resistant fish pathogens can cause significant economic loss to fish farmers. Since 2012, florfenicol has become an approved drug for treating both septicemia and columnaris diseases in freshwater fish. Due to the limited drug options available for aquaculture, the impact of the therapeutical florfenicol treatment on the microbiota landscape as well as the resistome present in the aquaculture farm environment needs to be evaluated. RESULTS: Time-series metagenomic analyses were conducted to the aquatic microbiota present in the tank-based catfish production systems, in which catfish received standard therapeutic 10-day florfenicol treatment following the federal veterinary regulations. Results showed that the florfenicol treatment shifted the structure of the microbiota and reduced the biodiversity of it by acting as a strong stressor. Planctomycetes, Chloroflexi, and 13 other phyla were susceptible to the florfenicol treatment and their abundance was inhibited by the treatment. In contrast, the abundance of several bacteria belonging to the Proteobacteria, Bacteroidetes, Actinobacteria, and Verrucomicrobia phyla increased. These bacteria with increased abundance either harbor florfenicol-resistant genes (FRGs) or had beneficial mutations. The florfenicol treatment promoted the proliferation of florfenicol-resistant genes. The copy number of phenicol-specific resistance genes as well as multiple classes of antibiotic-resistant genes (ARGs) exhibited strong correlations across different genetic exchange communities (p < 0.05), indicating the horizontal transfer of florfenicol-resistant genes among these bacterial species or genera. Florfenicol treatment also induced mutation-driven resistance. Significant changes in single-nucleotide polymorphism (SNP) allele frequencies were observed in membrane transporters, genes involved in recombination, and in genes with primary functions of a resistance phenotype. CONCLUSIONS: The therapeutical level of florfenicol treatment significantly altered the microbiome and resistome present in catfish tanks. Both intra-population and inter-population horizontal ARG transfer was observed, with the intra-population transfer being more common. The oxazolidinone/phenicol-resistant gene optrA was the most prevalent transferred ARG. In addition to horizontal gene transfer, bacteria could also acquire florfenicol resistance by regulating the innate efflux systems via mutations. The observations made by this study are of great importance for guiding the strategic use of florfenicol, thus preventing the formation, persistence, and spreading of florfenicol-resistant bacteria and resistance genes in aquaculture.

The two channel catfish intelectin genes exhibit highly differential patterns of tissue expression and regulation after infection with Edwardsiella ictaluri.

Intelectins (IntL) are Ca(2+)-dependent secretory glycoproteins that play a role in the innate immune response. The mammalian IntL is also known as lactoferrin receptor (LfR) that is involved in iron metabolism. The objective of this study was to characterize the intelectin genes in both channel catfish and blue catfish, to determine their genomic organization and copy numbers, to determine their patterns of tissue expression, and to establish if they are involved in defense responses of catfish after bacterial infection. Two types of IntL genes have been identified from catfish, and IntL2 was completely sequenced. The genomic structure and organization of IntL2 were similar to those of the mammalian species and of zebrafish and grass carp, but orthologies cannot be established with mammalian IntL genes. The IntL genes are highly conserved through evolution. Sequence analysis also indicated the presence of the fibrinogen-related domain in the catfish IntL genes, suggesting their structural conservations. Phylogenetic analysis suggested the presence of at least two prototypes of IntL genes in teleosts, but only one in mammals. The catfish IntL genes exhibited drastically different patterns of expression as compared to those of the mammalian species, or even with the grass carp gene. The catfish IntL1 gene is widely expressed in various tissues, whereas the channel catfish IntL2 gene was mainly expressed in the liver. While the catfish IntL1 is constitutively expressed, the catfish IntL2 was drastically induced by intraperitoneal injection of Edwardsiella ictaluri and/or iron dextran. Such induction was most dramatic when the fish were treated with both the bacteria and iron dextran. While IntL1 was expressed in all leukocyte cell lines, no expression of IntL2 was detected in any of the leukocyte cell lines, suggesting that the up-regulated channel catfish IntL2 expression after bacterial infection may be a consequence of the initial immune response, and/or a downstream immune response rather than a part of the primary immune responses.

Expression analysis of the acute phase response in channel catfish (Ictalurus punctatus) after infection with a Gram-negative bacterium.

The acute phase response (APR) is a set of metabolic and physiological reactions occurring in the host in response to tissue infection or injury and is a crucial component of the larger innate immune response. The APR is best characterized by dramatic changes in the concentration of a group of plasma proteins known as acute phase proteins (APPs) which are synthesized in the liver and function in a wide range of immunity-related activities. Utilizing a new high-density in situ oligonucleotide microarray, we have evaluated the APR in channel catfish liver following infection with Edwardsiella ictaluri, a bacterial pathogen that causes enteric septicemia of catfish. Our catfish microarray design (28K) builds upon a previous 19K channel catfish array by adding recently sequenced immune transcripts from channel catfish along with 7159 unique sequences from closely related blue catfish. The analysis of microarray results using a traditional 2-fold change in gene expression cutoff and a 10% false-discovery rate revealed a well-developed APR in catfish, with particularly high upregulation (>50-fold) of genes involved in iron homeostasis (i.e. intelectin, hemopexin, haptoglobin, ferritin, and transferrin). Other classical APP genes upregulated greater than 2-fold included coagulation factors, proteinase inhibitors, transport proteins, and complement components. Upregulation of the majority of the complement cascade was observed including the membrane attack complex components and complement inhibitors. A number of pathogen recognition receptors (PRRs) and chemokines were also differentially expressed in the liver following infection. Independent testing of a selection of differentially expressed genes with real-time RT-PCR confirmed microarray results.

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.

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.

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.

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.

Evasion of mucosal defenses during Aeromonas hydrophila infection of channel catfish (Ictalurus punctatus) skin.

The mucosal surfaces of fish serve as the first line of defense against the myriad of aquatic pathogens present in the aquatic environment. The immune repertoire functioning at these interfaces is still poorly understood. The skin, in particular, must process signals from several fronts, sensing and integrating environmental, nutritional, social, and health cues. Pathogen invasion can disrupt this delicate homeostasis with profound impacts on signaling throughout the organism. Here, we investigated the transcriptional effects of virulent Aeromonas hydrophila infection in channel catfish skin, Ictalurus punctatus. We utilized a new 8 × 60 K Agilent microarray for catfish to examine gene expression profiles at critical early timepoints following challenge--2 h, 8 h, and 12 h. Expression of a total of 2,168 unique genes was significantly perturbed during at least one timepoint. We observed dysregulation of genes involved in antioxidant, cytoskeletal, immune, junctional, and nervous system pathways. In particular, A. hydrophila infection rapidly altered a number of potentially critical lectins, chemokines, interleukins, and other mucosal factors in a manner predicted to enhance its ability to adhere to and invade the catfish host.