Authors: Allison L Wise, Benjamin R LaFrentz, Anita M Kelly...

and are pervasive bacterial pathogens associated with significant losses in catfish aquaculture. Bacterial coinfections have the potential to increase outbreak severity and can worsen on-farm mortality. A preliminary assessment of in vivo bacterial coinfection with (S97-773) and (ALG-00-530) was conducted using juvenile channel catfish (). Catfish were divided into five treatment groups: (1) mock control; (2) full dose (immersion; 5.4 × 10 CFU mL); (3) full dose (immersion; 3.6 × 10 CFU mL); (4) half dose (immersion; 2.7 × 10 CFU mL) followed by half dose (immersion; 1.8 × 10 CFU mL); and (5) half dose followed by half dose . In the coinfection challenges, the second inoculum was delivered 48 h after the initial exposure. At 21 days post-challenge (DPC), the single dose infection yielded a cumulative percent mortality (CPM) of 90.0 ± 4.1%, compared with 13.3 ± 5.9% in the group. Mortality patterns in coinfection challenges mimicked the single dose challenge, with CPM of 93.3 ± 5.4% for fish initially challenged with followed by , and 93.3 ± 2.7% for fish exposed to and subsequently challenged with . Despite similarities in the final CPM within the coinfection groups, the onset of peak mortality was delayed in fish exposed to first but was congruent with mortality trends in the challenge. Catfish exposed to in both the single and coinfected treatments displayed increased serum lysozyme activity at 4-DPC ( < 0.001). Three pro-inflammatory cytokines (, , ) were evaluated for gene expression, revealing an increase in expression at 7-DPC in all exposed treatments ( < 0.05). These data enhance our understanding of the dynamics of and coinfections in US farm-raised catfish.

PubMed: 36986384
DOI: 10.3390/pathogens12030462

Authors: Shamima Islam, Munshi Mustafiz Riman, Shahnewaj Mannan...

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.

Topics: Animals; Edwardsiella ictaluri; Escherichia coli; Enterobacteriaceae Infections; Plasmids; Anti-Bacterial Agents; Tetracycline; Catfishes; Drug Resistance, Microbial

PubMed: 36944411
DOI: 10.1016/j.jgar.2023.03.005

Authors: Safak Kalindamar, Hossam Abdelhamed, Adef O Kordon...

is a Gram-negative facultative intracellular pathogen causing enteric septicemia of catfish (ESC), a devastating disease resulting in significant economic losses in the U.S. catfish industry. Bacterial secretion systems are involved in many bacteria's virulence, and Type VI Secretion System (T6SS) is a critical apparatus utilized by several pathogenic Gram-negative bacteria. strain 93-146 genome has a complete T6SS operon with 16 genes, but the roles of these genes are still not explored. In this research, we aimed to understand the roles of two hemolysin co-regulated family proteins, Hcp1 (EvpC) and Hcp2. To achieve this goal, single and double mutants (ΔΔ, and ΔΔ) were generated and characterized. Catfish peritoneal macrophages were able to kill Δ better than ΔΔΔ, and wild-type (WT). The attachment of Δ and ΔΔ to ovary cells significantly decreased compared to WT whereas the cell invasion rates of these mutants were the same as that of WT. Mutants exposed to normal catfish serum showed serum resistance. The fish challenges demonstrated that Δ and ΔΔ were attenuated completely and provided excellent protection against WT infection in catfish fingerlings. Interestingly, Δ caused higher mortality than that of WT in catfish fingerlings, and severe clinical signs were observed. Although fry were more susceptible to vaccination with Δ and ΔΔ, their attenuation and protection were significantly higher compared to WT and sham groups, respectively. Taken together, our data indicated that () is involved in virulence in catfish while is involved in adhesion to epithelial cells and survival inside catfish macrophages.

PubMed: 34150898
DOI: 10.3389/fvets.2021.681609

Authors: James T Tuttle, Timothy J Bruce, Ian A E Butts...

Two prevalent bacterial diseases in catfish aquaculture are enteric septicemia of catfish and columnaris disease caused by and , respectively. Chronic and recurring outbreaks of these bacterial pathogens result in significant economic losses for producers annually. Determining if these pathogens can persist within sediments of commercial ponds is paramount. Experimental persistence trials (PT) were conducted to evaluate the persistence of and in pond sediments. Twelve test chambers containing 120 g of sterilized sediment from four commercial catfish ponds were inoculated with either (S97-773) or (ALG-00-530) and filled with 8 L of disinfected water. At 1, 2, 4-, 6-, 8-, and 15-days post-inoculation, 1 g of sediment was removed, and colony-forming units (CFU) were enumerated on selective media using 6 × 6 drop plate methods. population peaked on Day 3 at 6.4 ± 0.5 log CFU g. Correlation analysis revealed no correlation between the sediment physicochemical parameters and log CFU g. However, no viable colonies were recovered after two PT attempts. Future studies to improve understanding of pathogenesis and persistence, and potential persistence in pond bottom sediments are needed.

PubMed: 37513718
DOI: 10.3390/pathogens12070871

Review

Authors: Thirukanthan Chandra Segaran, Mohamad Nor Azra, Rumeaida Mat Piah...

This study aims to elucidate the evolution of catfish research publications over recent decades, identify emerging research clusters, examine keyword patterns, determine major contributors (including authors, organizations, and funding agencies), and analyze their collaborative networks and citation bursts on a global scale. The USA, Brazil, China, and India collectively contribute approximately 67% of the total catfish research publications, with a marked increase in prevalence since 2016. The most frequently occurring and dominant keywords are "channel catfish" and "responses," respectively. Intriguingly, our findings reveal 28 distinct article clusters, with prominent clusters including "yellow catfish," "channel catfish", "pectoral girdle," "African catfish", "Rio Sao Francisco basin," "," and "temperature mediated". Concurrently, keyword clustering generates seven main clusters: "new species", "growth performance", "heavy metal", "gonadotropin-releasing", "essential oil", and "olfactory receptor". This study further anticipates future research directions, offering fresh perspectives on the catfish literature landscape. To the best of our knowledge, this is the first article to conduct a comprehensive mapping review of catfish research publications worldwide.

PubMed: 37810135
DOI: 10.1016/j.heliyon.2023.e20081

Authors: Matthew L Rogge, Lidiya Dubytska, Tae Sung Jung...

We compared Edwardsiella ictaluri from striped catfish in Vietnam with US channel catfish isolates. Biochemical analyses and sequencing of the 16S rRNA gene confirmed that the Vietnamese isolates were E. ictaluri. Comparison using rep-PCR fingerprinting demonstrated no significant differences between the isolates, but plasmid analysis indicated that the Vietnamese isolates grouped into 4 plasmid profiles, each different from the typical pEI1 and pEI2 plasmid profile found in the US isolates. Sequencing plasmids representative of the 4 profiles indicated that all contained derivatives of the E. ictaluri plasmid pEI1, whereas only 1 contained a plasmid derivative of the E. ictaluri plasmid pEI2. The pEI2 encoded type III secretion effector, EseI, and its chaperone, EscD, were found to be present on the chromosome in isolates lacking a pEI2 derivative. In addition, 1 isolate carried a 5023 bp plasmid that does not have homology to either pEI1 or pEI2. Furthermore, Vietnamese isolates were PCR positive for the type III and type VI secretion system genes esrC and evpC, respectively, and the urease enzyme, but were PCR-negative for the putative type IV secretion system gene virD4. A monoclonal antibody against the lipopolysaccharide of E. ictaluri ATCC 33202 did not react with the Asian isolates or with the more recent US isolates. Antibiotic resistance patterns were variable and did not correlate to the presence of any particular plasmid profile. Finally, the Vietnamese isolates were avirulent and had a significantly reduced capacity for intracellular replication within head-kidney-derived channel catfish macrophages.

Topics: Animals; Anti-Bacterial Agents; Aquaculture; DNA Fingerprinting; Drug Resistance, Bacterial; Edwardsiella ictaluri; Enterobacteriaceae Infections; Fish Diseases; Gene Expression Regulation, Bacterial; Genome, Bacterial; Ictaluridae; Plasmids; Specific Pathogen-Free Organisms; United States; Vietnam; Virulence

PubMed: 24062549
DOI: 10.3354/dao02620

Comparative Study

Authors: Hasan C Tekedar, Jochen Blom, Safak Kalindamar...

and 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 93-146 and  C07-087 genomes were evaluated by applying comparative genomics analysis. All available complete (10) and non-complete (19) genomes from five species were also included in a systematic analysis. Average nucleotide identity and core-genome phylogenetic tree analyses indicated that the five species were separated from each other. Pan-/core-genome analyses for the 29 strains from the five species showed that genus members have 9474 genes in their pan genome, while the core genome consists of 1421 genes. Orthology cluster analysis showed that and  genomes have the greatest number of shared clusters. However, and  also have unique features; for example, the genome encodes urease enzymes and cytochrome o ubiquinol oxidase subunits, whereas  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 93-146 and three other genomes encode a type IV secretion system (T4SS), whereas none of the  genomes encode this system. Additionally, the  C07-087 genome encodes two different type VI secretion systems. genomes tend to encode more insertion elements, phage regions and genomic islands than . We speculate that the T4SS could contribute to the increased number of mobilome elements in compared to . Two of the  genomes encode full CRISPR-Cas regions, whereas none of the genomes encode Cas proteins. Overall, comparison of the and  genomes reveals unique features and provides new insights on pathogenicity that may reflect the host adaptation of the two species.

Topics: Animals; Catfishes; Edwardsiella; Edwardsiella ictaluri; Enterobacteriaceae Infections; Fish Diseases; Genome, Bacterial; Genomics; Phylogeny

PubMed: 32108566
DOI: 10.1099/mgen.0.000322

Authors: Safak Kalindamar, Jingjun Lu, Hossam Abdelhamed...

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.

Topics: Animals; Bacterial Vaccines; Computational Biology; DNA Transposable Elements; Edwardsiella ictaluri; Enterobacteriaceae Infections; Fish Diseases; Gene Deletion; Genome, Bacterial; Ictaluridae; Mutagenesis; Mutation; Phenotype; Vaccines, Attenuated; Virulence

PubMed: 30849940
DOI: 10.1186/s12866-019-1429-3

Authors: Min Zhang, Li Sun

Edwardsiella ictaluri is a Gram-negative bacterium and the causative agent of enteric septicemia of catfish. In this study, we examined the expression and function of the LuxS from a pathogenic E. ictaluri strain, 1901. J901 was found to produce autoinducer 2 (AI-2) activity that maximized at mid-logarithmic phase and was enhanced by glucose and repressed by high temperature. Consistently, a luxS gene (luxSEi) was identified in J901, whose expression was regulated by cell density, glucose, and temperature in a manner similar to that observed with AI-2 activity. Further analysis showed that LuxSEi is a biologically active AI-2 synthase that was able to complement the luxS-defective phenotype of Escherichia coli DH5alpha. To examine the functional importance of LuxSEi, a genetically modified variant of J901, J901Ri, was constructed, in which luxSEi, expression was blocked by RNA interference. Compared to the wild type, J901Ri was (i) reduced in AI-2 activity to a level of 59% of that of the wild type; (ii) impaired in both planktonic and biofilm growth; (iii) significantly attenuated in the ability to infect cultured fish cells and to cause mortality in infected fish; (iv) unable to induce the expression of certain virulence-associated genes. Addition of exogenous AI-2 failed to rescue the growth defect of J901Ri as free-living cells but restored biofilm production and the expression of virulence genes to levels comparable to those of the wild type. Taken together, these results indicate that LuxSEi is a functional AI-2 synthase that is required for optimal cellular growth and host infection.

Topics: Animals; Bacterial Proteins; Biofilms; Carbon-Sulfur Lyases; Cell Line; Edwardsiella ictaluri; Enterobacteriaceae Infections; Fish Diseases; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genome, Bacterial; Molecular Sequence Data; Quorum Sensing; Time Factors; Virulence; Zebrafish

PubMed: 23484409
DOI: No ID Found

Authors: Javier Santander, Wei Xin, Zhao Yang...

asdA mutants of gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd(+) plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd(+) plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd(+) expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd(+) vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd(+) plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Aspartate-Semialdehyde Dehydrogenase; Catfishes; Diaminopimelic Acid; Edwardsiella ictaluri; Fishes; Genetic Complementation Test; Molecular Sequence Data; Mutation; Plasmids; Salmonella; Sequence Homology, Amino Acid; Vaccines, Attenuated; Vaccines, Synthetic

PubMed: 21209920
DOI: 10.1371/journal.pone.0015944