Characterization of Type VI secretion system in Edwardsiella ictaluri.

Edwardsiella ictaluri is a Gram-negative facultative intracellular fish pathogen causing enteric septicemia of catfish (ESC). While various secretion systems contribute to E. ictaluri virulence, the Type VI secretion system (T6SS) remains poorly understood. In this study, we constructed 13 E. ictaluri T6SS mutants using splicing by overlap extension PCR and characterized them, assessing their uptake and survival in channel catfish (Ictalurus punctatus) peritoneal macrophages, attachment and invasion in channel catfish ovary (CCO) cells, in vitro stress resistance, and virulence and efficacy in channel catfish. Among the mutants, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO exhibited reduced replication inside peritoneal macrophages. EiΔevpM, EiΔevpN, and EiΔevpO showed significantly decreased attachment to CCO cells, while EiΔevpN and EiΔevpO also displayed reduced invasion of CCO cells (p < 0.05). Overall, T6SS mutants demonstrated enhanced resistance to oxidative and nitrosative stress in the nutrient-rich medium compared to the minimal medium. However, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO were susceptible to oxidative stress in both nutrient-rich and minimal medium. In fish challenges, EiΔevpD, EiΔevpE, EiΔevpG, EiΔevpJ, and EiΔevpK exhibited attenuation and provided effective protection against E. ictaluri wild-type (EiWT) infection in catfish fingerlings. However, their attenuation and protective efficacy were lower in catfish fry. These findings shed light on the role of the T6SS in E. ictaluri pathogenesis, highlighting its significance in intracellular survival, host cell attachment and invasion, stress resistance, and virulence. The attenuated T6SS mutants hold promise as potential candidates for protective immunization strategies in catfish fingerlings.

Synergistic infection of Edwardsiella ictaluri and Flavobacterium oreochromis in cage cultured tilapia (Oreochromis sp.).

Widespread distribution of a highly pathogenic Edwardsiella ictaluri strain in farmed tilapia in northern Vietnam has recently been reported. The subsequent investigation noticed a disease outbreak occurred at five nearby tilapia farms with floating cages, in which the clinical signs of both edwardsiellosis and columnaris diseases were observed on the same infected fish and caused 65% to 85% fish mortality. Naturally diseased fish (n = 109) were sampled from the five infected farms for bacterial identification and conducting challenge tests. The two bacteria Edwardsiella ictaluri and Flavobacterium oreochromis were identified by a combination of biochemical tests, PCR and 16SrRNA sequencing methods. Experimental challenge tests on Nile tilapia resulted in the median lethal dose (LD50 ) of E. ictaluri and F. oreochromis at 70 CFU/fish by intraperitoneal (i.p.) injection and 3.6 × 106 CFU/mL by immersion, respectively. The experimentally co-infected challenged fish exposed to LD50 doses resulted in 83% ± 6% mortality, with the infected fish exhibiting clinical signs of both edwardsiellosis and columnaris diseases, mimicking the naturally diseased fish. This finding suggests that the co-infection of E. ictaluri and F. oreochromis may interact in a synergistic manner, to enhance the overall severity of the infection and elevates the need for efficient methods to control both pathogens.

Characterisation and mobilisation of IncA/C plasmid-mediated antibiotic resistance in Edwardsiella ictaluri.

OBJECTIVES: Edwardsiella ictaluri is an important pathogen in farmed raised catfish. Recently, we showed that resistance to tetracycline and florfenicol in the E. ictaluri MS-17-156 strain isolated from channel catfish was facilitated by acquisition of a 135 kb plasmid (named pEIMS-171561). METHODS: We described the genetic structure of pEIMS-171561. Plasmid copy number and stability within E. ictaluri strain MS-17-156 was determined. We also investigated the in vitro and in vivo transferability of pEIMS-171561 using catfish as a model for in vivo transfer. RESULTS: pEIMS-171561 belonged to the IncA/C group and contained florfenicol efflux major facilitator superfamily (MFS) (floR), sulfonamides (sul2), and tetracycline efflux MFS (tetD) genes. The plasmid contained two conjugative transfer-associated regions and encoded six transposases and insertion sequences. In vitro conjugation experiments demonstrated that the IncA/C plasmid can transfer from E. ictaluri to Escherichia coli. The plasmid was stable in E. ictaluri without selection pressure for 33 days. We showed that pEIMS-171561 did not transfer from E. ictaluri MS-17-156 to endogenous microbiota in catfish. Moreover, we could not detect in vivo conjugal transfer of pEIMS-171561 from E. ictaluri to E. coli. Results from real-time PCR revealed upregulation of the floR gene in the intestines of catfish receiving florfenicol-medicated feed, compared with that in catfish receiving unmedicated feed. CONCLUSION: This study demonstrated that pEIMS-171561 did not disseminate from E. ictaluri to gut microbiota under selective pressure. This result suggests a limited role of the fish microbiota as a reservoir for this plasmid and for the spread of resistance.

The Attenuation Mechanism and Live Vaccine Potential of a Low-Virulence Edwardsiella ictaluri Strain Obtained by Rifampicin Passaging Culture.

The rifampicin-resistant strain E9-302 of Edwardsiella ictaluri strain 669 (WT) was generated by continuous passage on BHI agar plates containing increasing concentrations of rifampicin. E9-302 was attenuated significantly by 119 times to zebrafish Danio rerio compared to WT in terms of the 50% lethal dose (LD50). Zebrafish vaccinated with E9-302 via intraperitoneal (IP) injection at a dose of 1 × 103 CFU/fish had relative percentage survival (RPS) rates of 85.7% when challenged with wild-type E. ictaluri via IP 14 days post-vaccination (dpv). After 14 days of primary vaccination with E9-302 via immersion (IM) at a dose of 4 × 107 CFU/ml, a booster IM vaccination with E9-302 at a dose of 2 × 107 CFU/ml exhibited 65.2% RPS against challenge with wild-type E. ictaluri via IP 7 days later. These results indicated that the rifampicin-resistant attenuated strain E9-302 had potential as a live vaccine against E. ictaluri infection. A previously unreported amino acid site change at position 142 of the RNA polymerase (RNAP) ß subunit encoded by the gene rpoB associated with rifampicin resistance was identified. Analysis of the whole-genome sequencing results revealed multiple missense mutations in the virulence-related genes esrB and sspH2 in E9-302 compared with WT, and a 189 bp mismatch in one gene, whose coding product was highly homologous to glycosyltransferase family 39 protein. This study preliminarily explored the molecular mechanism underlying the virulence attenuation of rifampicin-resistant strain E9-302 and provided a new target for the subsequent study of the pathogenic mechanism of E. ictaluri.

Evaluation of Edwardsiella piscicida basS and basR mutants as vaccine candidates in catfish against edwardsiellosis.

Catfish farming is the largest aquaculture industry in the United States and an important economic driver in several southeastern states. Edwardsiella piscicida is a Gram-negative pathogen associated with significant losses in catfish aquaculture. Several Gram-negative bacteria use the BasS/BasR two-component system (TCS) to adapt to environmental changes and the host immune system. Currently, the role of BasS/BasR system in E. piscicida virulence has not been characterized. In the present study, two mutants were constructed by deleting the basS and basR genes in E. piscicida strain C07-087. Both mutant strains were characterized for virulence and immune protection in catfish hosts. The EpΔbasS and EpΔbasR mutants were more sensitive to acidic environments and produced significantly less biofilm than the wild-type. In vivo studies in channel catfish (Ictalurus punctatus) revealed that both EpΔbasS and EpΔbasR were significantly attenuated compared with the parental wild-type (3.57% and 4.17% vs. 49.16% mortalities). Moreover, there was significant protection, 95.2% and 92.3% relative percent survival (RPS), in channel catfish vaccinated with EpΔbasS and EpΔbasR against E. piscicida infection. Protection in channel catfish was associated with a significantly higher level of antibodies and upregulation of immune-related genes (IgM, IL-8 and CD8-α) in channel catfish vaccinated with EpΔbasS and EpΔbasR strains compared with non-vaccinated fish. Hybrid catfish (channel catfish ♀ × blue catfish ♂) challenges demonstrated long-term protection against subsequent challenges with E. piscicida and E. ictaluri. Our findings demonstrate BasS and BasR contribute to acid tolerance and biofilm formation, which may facilitate E. piscicida survival in harsh environments. Further, our results show that EpΔbasS and EpΔbasR mutants were safe and protective in channel catfish fingerlings, although their virulence and efficacy in hybrid catfish warrant further investigation. These data provide information regarding an important mechanism of E. piscicida virulence, and it suggests EpΔbasS and EpΔbasR strains have potential as vaccines against this emergent catfish pathogen.

Widespread presence of a highly virulent Edwardsiella ictaluri strain in farmed tilapia, Oreochromis spp.

Edwardsiella ictaluri is an emerging bacterial pathogen that affects farmed tilapia (Oreochromis spp.). This study reports the widespread presence of E. ictaluri in farmed tilapia in Vietnam. Among 26 disease outbreaks from nine provinces in Northern Vietnam during 2019-2021, 19 outbreaks originated from imported seeds, while outbreaks in seven farms were from domestic sources. Clinically sick fish showed the appearance of numerous white spots in visceral organs, and accumulative mortality reached 30%-65%. A total of 26 representative bacterial isolates recovered from 26 disease outbreaks were identified as E. ictaluri based on a combination of phenotypic tests, genus- and species-specific polymerase chain reaction assays, 16S rRNA and gyrB sequencing, and phylogenetic analysis. All isolates harboured the same virulence gene profiles esrC+ , evpC+ , ureA-C+ , eseI- , escD- and virD4- . Antimicrobial susceptibility tests revealed that 80.8%-100% of isolates were multidrug resistant, with resistance to 4-8 antimicrobials in the groups of penicillin, macrolides, sulfonamides, amphenicols and glycopeptides. The experimental challenge successfully induced disease that mimicked natural infection. The median lethal doses (LD50 ) of the tested isolates (n = 4) were 42-61 colony forming units/fish, indicating their extremely high virulence. This emerging pathogen is established and has spread to various geographical locations, causing serious impacts on farmed tilapia in northern Vietnam. It is likely that this pathogen will continue to spread through contaminated stocks (both imported and domestic sources) and persist. Thus, increased awareness, combined with biosecurity measures and emergent vaccination programs is essential to mitigate the negative impact of this emerging disease on the tilapia farming industry.

Comparative genomics of the fish pathogens Edwardsiella ictaluri 93-146 and Edwardsiella piscicida C07-087.

Edwardsiella ictaluri and Edwardsiella piscicida are important fish pathogens affecting cultured and wild fish worldwide. To investigate the genome-level differences and similarities between catfish-adapted strains in these two species, the complete E. ictaluri 93-146 and E. piscicida C07-087 genomes were evaluated by applying comparative genomics analysis. All available complete (10) and non-complete (19) genomes from five Edwardsiella species were also included in a systematic analysis. Average nucleotide identity and core-genome phylogenetic tree analyses indicated that the five Edwardsiella species were separated from each other. Pan-/core-genome analyses for the 29 strains from the five species showed that genus Edwardsiella members have 9474 genes in their pan genome, while the core genome consists of 1421 genes. Orthology cluster analysis showed that E. ictaluri and E. piscicida genomes have the greatest number of shared clusters. However, E. ictaluri and E. piscicida also have unique features; for example, the E. ictaluri genome encodes urease enzymes and cytochrome o ubiquinol oxidase subunits, whereas E. piscicida genomes encode tetrathionate reductase operons, capsular polysaccharide synthesis enzymes and vibrioferrin-related genes. Additionally, we report for what is believed to be the first time that E. ictaluri 93-146 and three other E. ictaluri genomes encode a type IV secretion system (T4SS), whereas none of the E. piscicida genomes encode this system. Additionally, the E. piscicida C07-087 genome encodes two different type VI secretion systems. E. ictaluri genomes tend to encode more insertion elements, phage regions and genomic islands than E. piscicida. We speculate that the T4SS could contribute to the increased number of mobilome elements in E. ictaluri compared to E. piscicida. Two of the E. piscicida genomes encode full CRISPR-Cas regions, whereas none of the E. ictaluri genomes encode Cas proteins. Overall, comparison of the E. ictaluri and E. piscicida genomes reveals unique features and provides new insights on pathogenicity that may reflect the host adaptation of the two species.

Transposon mutagenesis and identification of mutated genes in growth-delayed Edwardsiella ictaluri.

BACKGROUND: Edwardsiella ictaluri is a Gram-negative facultative intracellular anaerobe and the etiologic agent of enteric septicemia of channel catfish (ESC). To the catfish industry, ESC is a devastating disease due to production losses and treatment costs. Identification of virulence mechanisms of E. ictaluri is critical to developing novel therapeutic approaches for the disease. Here, we report construction of a transposon insertion library and identification of mutated genes in growth-delayed E. ictaluri colonies. We also provide safety and efficacy of transposon insertion mutants in catfish. RESULTS: An E. ictaluri transposon insertion library with 45,000 transposants and saturating 30.92% of the TA locations present in the E. ictaluri genome was constructed. Transposon end mapping of 250 growth-delayed E. ictaluri colonies and bioinformatic analysis of sequences revealed 56 unique E. ictaluri genes interrupted by the MAR2xT7 transposon, which are involved in metabolic and cellular processes and mostly localized in the cytoplasm or cytoplasmic membrane. Of the 56 genes, 30 were associated with bacterial virulence. Safety and vaccine efficacy testing of 19 mutants showed that mutants containing transposon insertions in hypothetical protein (Eis::004), and Fe-S cluster assembly protein (IscX, Eis::039), sulfurtransferase (TusA, Eis::158), and universal stress protein A (UspA, Eis::194) were safe and provided significant protection (p < 0.05) against wild-type E. ictaluri. CONCLUSIONS: The results indicate that random transposon mutagenesis causing growth-delayed phenotype results in identification bacterial virulence genes, and attenuated strains with transposon interrupted virulence genes could be used as vaccine to activate fish immune system.

Development and potential use of an Edwardsiella ictaluri wzz mutant as a live attenuated vaccine against enteric septicemia in Pangasius hypophthalmus (Tra catfish).

Edwardsiella ictaluri is a causative agent of enteric septicemia of catfish (ESC), a seriously lethal disease in Vietnamese catfish (Pangasius hypophthalmus). A safe and effective vaccine against ESC is currently an urgent demand due to antibiotic overuse in pangasius farms has led to an alarming antimicrobial resistance. In this study, two E. ictaluri wzzE mutants (WzM-L3, deficient in a 1038bp-entire wzzE gene and WzM-S3, a 245bp-partial deletion of wzzE) were developed and their protection efficiacy was evaluated in hatched pangasius against ESC by immersion vaccination. As comparing to the high virulent wild-type strain who caused 73.33% of death on pangasius fingerlings immersed at 7.1 × 106 CFU ml-1, both mutants showed extremely low mortality rates at 3.33% (WzM-S3) and 0% (WzM-L3) on pangasius fingerlings immersed at high concentration of 1.5 × 107 CFU mL-1 and 9.7 × 106 CFU ml-1, respectively. Interestingly, both WzM-S3 and WzM-L3 had a remarkably high protection against ESC, as RPS % were found at 89.29% and 90%, respectively. The mutant WzM-L3 is a potential live attenuated vaccine against ESC in Vietnamese catfish farms with good protection and simple practice.

The virulence and immune protection of Edwardsiella ictaluri HemR mutants in catfish.

Edwardsiella ictaluri is a Gram-negative facultative intracellular rod, causing enteric septicemia of catfish (ESC). Several heme uptake systems have been described in bacterial pathogens, most of which involve outer membrane proteins (OMPs). We have shown recently that heme/hemoglobin receptor family protein (HemR) is significantly up-regulated in E. ictaluri under iron-restricted conditions. In this work, our goal was to construct E. ictaluri HemR mutants and assess their virulence and immune protection potentials in catfish. To accomplish this, an in-frame deletion mutant (EiΔhemR) was constructed, and its virulence and immune protection were determined in catfish fingerlings and fry. The results indicated that the EiΔhemR was attenuated completely in catfish fingerlings, but it was virulent in 14 day-old catfish fry. To increase the attenuation of EiΔhemR in fry, we introduced frdA and sdhC gene deletions to the mutant, yielding two double (EiΔhemRΔfrdA and EiΔhemRΔsdhC) and one triple (EiΔhemRΔfrdAΔsdhC) mutants. Results indicated that two double HemR mutants did not exhibit increased attenuation, but the triple HemR mutant showed significantly less virulence and high protection in fry (p < 0.05). Histological examination of fry tissues vaccinated with the triple mutant displayed similar inflammation to that of wild-type infected fry, but much less necrosis and far fewer bacteria were observed. Immunohistochemistry (IHC) result indicated fewer numbers of bacteria around blood vessel and in the hematopoietic tissue in fry infected with triple mutant compared to control group infected with E. ictaluri wild-type. Our data indicated that EiΔhemR was safe and protective in catfish fingerlings, while EiΔhemRΔfrdAΔsdhC was much safer in catfish fry.

Improving safety of a live attenuated Edwardsiella ictaluri vaccine against enteric septicemia of catfish and evaluation of efficacy.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of channel catfish (ESC). Our recent work indicated that tricarboxylic acid cycle and one-carbon metabolism are critical pathways for E. ictaluri virulence. Although single and double gene deletions in these pathways resulted in safe and efficacious vaccines for use in catfish fingerlings, vaccine trials in catfish fry showed safety concerns. Therefore, we aimed to improve the safety of these mutants by constructing two triple mutant combinations. ESC-NDKL1 (ΔgcvPΔsdhCΔfrdA) was constructed by introducing an in-frame deletion of frdA in a gcvP-sdh mutant. ESC-NDKL2 (ΔgcvPΔsdhCΔmdh) was constructed in a similar manner. ESC-NDKL1 strain was a better vaccine candidate compared to ESC-NDKL2, providing better safety and efficacy in catfish fry and catfish fingerlings. Field trials in earthen ponds under three vaccination conditions showed that survival was significantly higher in catfish vaccinated with ESC-NDKL1 by immersion at the fry stage, oral vaccination in ponds, and fry immersion-pond oral combination (86.74%, 81.67%, and 95.22%, respectively) compared to sham-vaccinated (42.75%), and Aquavac-ESC fry immersion vaccinated (61.51%) catfish. Our findings indicate that ESC-NDKL1 is a good candidate for further development as a vaccine for ESC.

Edwardsiella ictaluri infection in Pangasius catfish imported from West Bengal into the Southern Caribbean.

In response to a mortality event, seven Pangasius catfish (Pangasianodon hypophthalmus) were submitted to the University of the West Indies, School of Veterinary Medicine, Trinidad and Tobago, for diagnostic evaluation. These fish were part of a consignment that arrived from Kolkata two weeks earlier. Fish presented with perianal haemorrhage and blister-like swellings on the skin which ruptured to leave ulcers. Edwardsiella ictaluri was consistently recovered from the brain and skin. Repetitive sequence-mediated PCR analysis revealed genetic fingerprints consistent with E. ictaluri isolates from farm-raised channel catfish in Mississippi, USA. Plasmid analysis of the case isolates identified two unique plasmids that differ slightly in conformation and content from the pEI1 and pEI2 plasmids described for E. ictaluri from other fish hosts. The case isolates were also PCR negative for several E. ictaluri virulence factors. The biological implications of these genetic differences are unclear and warrant further study. This is the first report and documentation of E. ictaluri infection in Trinidad and Tobago, suggesting the pathogen may have been introduced concurrently with the importation of fish. This report emphasizes the importance of adequate health screenings of imported lots to minimize the threat of introducing E. ictaluri to non-endemic areas.

Ferric hydroxamate uptake system contributes to Edwardsiella ictaluri virulence.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia in fish, particularly in channel catfish. Ferric iron is an essential micronutrient for bacterial survival, and some bacterial pathogens use secreted hydroxamate-type siderophores to chelate iron in host tissues. Siderophore-iron complexes are taken up by these bacteria via the ferric hydroxamate uptake (Fhu) system. In E. ictaluri, the Fhu system consists of fhuC, fhuD, fhuB, and fhuA genes. However, the importance of the Fhu system in E. ictaluri virulence has not been investigated completely. Here, we present construction of E. ictaluri fhuD and fhuB mutants (EiΔfhuD and EiΔfhuB) by in-frame gene deletion and evaluation of the mutants' virulence and immunogenicity in channel catfish fingerlings and fry. Immersion challenges showed that EiΔfhuD was not significantly attenuated (p < 0.05) in catfish fingerlings, whereas EiΔfhuB was significantly attenuated (p < 0.01). Catfish fingerlings immunized with EiΔfhuD and EiΔfhuB showed 100% and 97.62% survival, respectively. Fry immersion challenges indicated EiΔfhuB was also significantly attenuated (p < 0.05) in two-week old fry compared to the wild-type (48.96% vs. 82.14% mortalities). The survival rate in the fry vaccinated with EiΔfhuB was significantly higher (p < 0.05) than that of non-vaccinated fry (96.77% vs. 21.42% survival). Our data indicates that the fhuB gene, but not the fhuD gene, contributes to E. ictaluri virulence.

Involvement of tolQ and tolR genes in Edwardsiella ictaluri virulence.

Edwardsiella ictaluri is a Gram-negative intracellular facultative pathogen causing enteric septicemia of channel catfish (ESC). The Tol system, consisting of four envelope proteins TolQ, TolR, TolA, and TolB, are required for colicin import and contributes to bacterial virulence in several pathogenic bacteria. However, the Tol system and its importance in E. ictaluri virulence have not been investigated. Here we present construction and evaluation of the E. ictaluri TolQ, TolR and TolQR mutants (EiΔtolQ, EiΔtolR, and EiΔtolQR). The Tol mutants were developed using in-frame gene deletion and their attenuation and vaccine efficacy were determined in catfish fingerlings. The EiΔtolQ, EiΔtolR, and EiΔtolQR mutants showed reduced virulence in catfish (28.93%, 19.70%, and 39.82% mortality, respectively) compared to wild type (46.91% mortality). Further, vaccination with these mutants protected catfish against subsequent wild-type infection. This study suggests that the Tol system contributes to E. ictaluri virulence in catfish.

Influence of lipopolysaccharide outer-core in the intrinsic resistance to antimicrobial peptides and virulence in Edwardsiella ictaluri.

The genus Edwardsiella consists of bacteria with an intrinsic resistance to cyclic cationic antimicrobial peptides (CAMPs). Edwardsiella ictaluri, a pathogen of the catfish (Ictalurus punctatus) and the causative agent of a systemic infection, is highly resistant to CAMPs. Previously, we determined that the oligo-polysaccharide (O-PS) of the lipopolysaccharide (LPS) does not play a role in the E. ictaluri CAMP resistance and an intact core-lipid A structure is necessary for CAMPs resistance. Here, we evaluated the influence of the outer-core in the CAMPs resistance and fish virulence. E. ictaluri wabG, a gene that encodes for the UDP-glucuronic acid transferase that links the lipid A-inner-core to the outer-core-oligopolysaccharides, was deleted. Deletion of ΔwabG caused a pleiotropic effect, influencing LPS synthesis, CAMPs resistance, growth, and biofilm formation. E. ictaluri ΔwabG was attenuated in zebrafish indicating the important role of LPS during fish pathogenesis. Also, we evaluated the inflammatory effects of wabG LPS in catfish ligated loop model, showing a decreased inflammatory effect at the gut level respects to the E. ictaluri wild type. We conclude that E. ictaluri CAMPs resistance is related to the molecules present in the LPS outer-core and that fish gut inflammation triggered by E. ictaluri is LPS dependent, reinforcing the hypothesis that fish gut recognizes LPS in an O-PS dependent fashion.

Comparison of Edwardsiella ictaluri isolates from different hosts and geographic origins.

The intraspecific variability of E. ictaluri isolates from different origins was investigated. Isolates were recovered from farm-raised catfish (Ictalurus punctatus) in Mississippi, USA, tilapia (Oreochromis niloticus) cultured in the Western Hemisphere and zebrafish (Danio rerio) propagated in Florida, USA. These isolates were phenotypically homologous and antimicrobial profiles were largely similar. Genetically, isolates possessed differences that could be exploited by repetitive-sequence-mediated PCR and gyrB sequence, which identified three distinct E. ictaluri genotypes: one associated with catfish, one from tilapia and a third from zebrafish. Plasmid profiles were also group specific and correlated with rep-PCR and gyrB sequences. The catfish isolates possessed profiles typical of those described for E. ictaluri isolates; however, plasmids from the zebrafish and tilapia isolates differed in both composition and arrangement. Furthermore, some zebrafish and tilapia isolates were PCR negative for several E. ictaluri virulence factors. Isolates were serologically heterogenous, as serum from a channel catfish exposed to a catfish isolate had reduced antibody activity to tilapia and zebrafish isolates. This work identifies three genetically distinct strains of E. ictaluri from different origins using rep-PCR, 16S, gyrB and plasmid sequencing, in addition to antimicrobial and serological profiling.

Inflammatory effects of Edwardsiella ictaluri lipopolysaccharide modifications in catfish gut.

Bacterial lipopolysaccharides (LPS) are structural components of the outer membranes of Gram-negative bacteria and also are potent inducers of inflammation in mammals. Higher vertebrates are extremely sensitive to LPS, but lower vertebrates, like fish, are resistant to their systemic toxic effects. However, the effects of LPS on the fish intestinal mucosa remain unknown. Edwardsiella ictaluri is a primitive member of the Enterobacteriaceae family that causes enteric septicemia in channel catfish (Ictalurus punctatus). E. ictaluri infects and colonizes deep lymphoid tissues upon oral or immersion infection. Both gut and olfactory organs are the primary sites of invasion. At the systemic level, E. ictaluri pathogenesis is relatively well characterized, but our knowledge about E. ictaluri intestinal interaction is limited. Recently, we observed that E. ictaluri oligo-polysaccharide (O-PS) LPS mutants have differential effects on the intestinal epithelia of orally inoculated catfish. Here we evaluate the effects of E. ictaluri O-PS LPS mutants by using a novel catfish intestinal loop model and compare it to the rabbit ileal loop model inoculated with Salmonella enterica serovar Typhimurium LPS. We found evident differences in rabbit ileal loop and catfish ileal loop responses to E. ictaluri and S. Typhimurium LPS. We determined that catfish respond to E. ictaluri LPS but not to S. Typhimurium LPS. We also determined that E. ictaluri inhibits cytokine production and induces disruption of the intestinal fish epithelia in an O-PS-dependent fashion. The E. ictaluri wild type and ΔwibT LPS mutant caused intestinal tissue damage and inhibited proinflammatory cytokine synthesis, in contrast to E. ictaluri Δgne and Δugd LPS mutants. We concluded that the E. ictaluri O-PS subunits play a major role during pathogenesis, since they influence the recognition of the LPS by the intestinal mucosal immune system of the catfish. The LPS structure of E. ictaluri mutants is needed to understand the mechanism of interaction.

Tissue persistence and vaccine efficacy of tricarboxylic acid cycle and one-carbon metabolism mutant strains of Edwardsiella ictaluri.

Edwardsiella ictaluri causes enteric septicemia in fish. Recently, we reported construction of E. ictaluri mutants with single and double gene deletions in tricarboxylic acid cycle (TCA) and one-carbon (C-1) metabolism. Here, we report the tissue persistence, virulence, and vaccine efficacy of TCA cycle (EiΔsdhC, EiΔfrdA, and EiΔmdh), C-1 metabolism (EiΔgcvP and EiΔglyA), and combination mutants (EiΔfrdAΔsdhC, EiΔgcvPΔsdhC, EiΔmdhΔsdhC, and EiΔgcvPΔglyA) in channel catfish. The tissue persistence study showed that EiΔsdhC, EiΔfrdA, EiΔfrdAΔsdhC, and EiΔgcvPΔsdhC were able to invade catfish and persist until 11 days post-infection. Vaccination of catfish fingerlings with all nine mutants provided significant (P<0.05) protection against subsequent challenge with the virulent parental strain. Vaccinated catfish fingerlings had 100% survival when subsequently challenged by immersion with wild-type E. ictaluri except for EiΔgcvPΔglyA and EiΔgcvP. Mutant EiΔgcvPΔsdhC was found to be very good at protecting catfish fry, as evidenced by 10-fold higher survival compared to non-vaccinated fish.

Effect of multiple mutations in tricarboxylic acid cycle and one-carbon metabolism pathways on Edwardsiella ictaluri pathogenesis.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of catfish (ESC). We have shown recently that tricarboxylic acid cycle (TCA) and one-carbon (C1) metabolism are involved in E. ictaluri pathogenesis. However, the effect of multiple mutations in these pathways is unknown. Here, we report four novel E. ictaluri mutants carrying double gene mutations in TCA cycle (EiΔmdhΔsdhC, EiΔfrdAΔsdhC), C1 metabolism (EiΔglyAΔgcvP), and both TCA and C1 metabolism pathways (EiΔgcvPΔsdhC). In-frame gene deletions were constructed by allelic exchange and mutants' virulence and vaccine efficacy were evaluated using in vivo bioluminescence imaging (BLI) as well as end point mortality counts in catfish fingerlings. Results indicated that all the double gene mutants were attenuated compared to wild-type (wt) E. ictaluri. There was a 1.39-fold average reduction in bioluminescence, and hence bacterial numbers, from all the mutants except for EiΔfrdAΔsdhC at 144 h post-infection. Vaccination with mutants was very effective in protecting channel catfish against subsequent infection with virulent E. ictaluri 93-146 strain. In particular, immersion vaccination resulted in complete protection. Our results provide further evidence on the importance of TCA and C1 metabolism pathways in bacterial pathogenesis.

Phenotypic traits, virulence-associated gene profile and genetic relatedness of Edwardsiella tarda isolates from Japanese eel Anguilla japonica in Korea.

UNLABELLED: Edwardsiella tarda is the predominant bacterium in farm-cultured eel in Korea. Here, we evaluated the heterogeneity of 37 E. tarda isolates derived from Japanese eel with various origins (olive flounder, common carp and ornamental fish) between 2003 and 2010. Regardless of origins, the biochemical characteristics of E. tarda isolates were homogenous except hydrogen sulfide production, citrate utilization and mannitol fermentation. Based on the phylogenetic analysis of 16S rRNA, E. tarda isolates could be classified into two subgroups and displayed a close relation with Edwardsiella ictaluri and Edwardsiella hosinae lineages, suggesting that the subgroup I has been a predominant type in the Jeonnam and Jeonbuk provinces. I-CeuI-based pulsed-field gel electrophoresis (PFGE) typing showed that the isolates from Japanese eels belonged to 11 pulsotypes, indicating that the presence of highly genomic diversity. Additionally, two isolates, ET-060 and ET-191, showed a high frequency of virulence genes (100%) and caused 90% and 60% mortality in Japanese eel, respectively. This finding suggests a substantial congruence of virulence gene profiles and pathogenicity. Our results demonstrate that the intraspecific diversity within E. tarda strains from Japanese eel has been in prior existence. SIGNIFICANCE AND IMPACT OF THE STUDY: Based on the biochemical characteristics, the phylogenetic property of the 16S rRNA gene and PFGE types of Edwardsiella tarda, we could identify the intraspecific diversity of isolates from Japanese eel, Anguilla japonica in Korea. In addition, this study describes the strong congruence of virulence-related genes and pathogenicity, suggesting that the virulence profile may be useful tool for prediction of pathogenicity.