Edwardsiella piscicida lacking the cyclic AMP receptor protein (Crp) is avirulent and immunogenic in fish.

Edwardsiella piscicida is a Gram-negative pathogen that generally causes lethal septicemia in marine and freshwater fish. We generated a E. piscicida CK216 Δcrp mutant to investigate various biological roles related to this organism, including pathogenesis. Lack of Crp in CK216 was demonstrated by immunoblotting using a Crp-specific antibody. Compared to the parental strain, the mutant exhibited changes in three biochemical phenotypes, including ornithine decarboxylation, citrate utilization, and H2S production. Complementation of crp deletion in trans rescued the phenotype of the parental strain. This study proved that hemolytic activity in E. piscicida is controlled by Crp. In addition, significantly reduced motility of E. piscicida CK216 was observed, which resulted from a lack of flagella synthesis. To examine the virulence in fish, E. piscicida cells were injected into the goldfish (Carassius auratus) via intraperitoneal route. The LD50 of CK216 was 9.25 × 108 CFU, while that of the CK108 parental strain was 9.24 × 105 CFU, attenuated 1000 fold in goldfish. Fish immunized with CK216 elicited IgM responses. Moreover, 80% of goldfish immunized with 1 × 106 CFU survived after administration of a lethal dose (1 × 107 CFU) of virulent E. piscicida CK41, suggesting the potential for E. piscicida CK216 to serve as a live attenuated vaccine in aquaculture.

Phylogenomic network and comparative genomics reveal a diverged member of the ΦKZ-related group, marine vibrio phage ΦJM-2012.

Bacteriophages are the largest reservoir of genetic diversity. Here we describe the novel phage ΦJM-2012. This natural isolate from marine Vibrio cyclitrophicus possesses very few gene contents relevant to other well-studied marine Vibrio phages. To better understand its evolutionary history, we built a mathematical model of pairwise relationships among 1,221 phage genomes, in which the genomes (nodes) are linked by edges representing the normalized number of shared orthologous protein families. This weighted network revealed that ΦJM-2012 was connected to only five members of the Pseudomonas ΦKZ-like phage family in an isolated network, strongly indicating that it belongs to this phage group. However, comparative genomic analyses highlighted an almost complete loss of colinearity with the ΦKZ-related genomes and little conservation of gene order, probably reflecting the action of distinct evolutionary forces on the genome of ΦJM-2012. In this phage, typical conserved core genes, including six RNA polymerase genes, were frequently displaced and the hyperplastic regions were rich in both unique genes and predicted unidirectional promoters with highly correlated orientations. Further, analysis of the ΦJM-2012 genome showed that segments of the conserved N-terminal parts of ΦKZ tail fiber paralogs exhibited evidence of combinatorial assortment, having switched transcriptional orientation, and there was recruitment and/or structural changes among phage endolysins and tail spike protein. Thus, this naturally occurring phage appears to have branched from a common ancestor of the ΦKZ-related groups, showing a distinct genomic architecture and unique genes that most likely reflect adaptation to its chosen host and environment.

Combination treatment against scuticociliatosis by reducing the inhibitor effect of mucus in olive flounder, Paralichthys olivaceus.

The olive flounder, Paralichthys olivaceus, is an economically important food fish in Japan and Korea. Scuticociliatosis is a major parasitic disease, and fatal infection with scuticociliates, or mixed infections with scuticociliates and other pathogenic agents (e.g., Vibrio spp.) cause severe mortalities in farmed olive flounders. To date, however, effective chemotherapeutic treatment of scuticociliatosis has only been reported at the in vitro level. In this study, we employed combination treatment, using benzalkonium chloride (to remove excess mucus from the body surface) and bronopol (to kill the parasites), to overcome the protective effect of mucus by some medicine to the scuticociliates. In the presence of the mucus mixture, the higher dose of bronopol (156 ppm) yielded morphologies and motilities similar to those of ciliates treated with the lower dose of bronopol (80 ppm) in the absence of mucus. We also investigated the in vivo effects of this treatment in field trials involving a total of 15,025 naturally infected flounders. We observed that short-term bath treatments with benzalkonium chloride (50 ppm) followed by bronopol (500 ppm) were effective, assessed by the relative percentage mortality (RPS) value. Thus, this study provides a notable therapeutic strategy by removing the mucus to treat scuticociliatosis in olive flounders at the aquaculture field level.

Role of sigma factor E in regulation of Salmonella Agf expression.

Expression of thin aggregative fimbriae (Agf) in Salmonella, which is responsible for bacterial cell adhesion to surfaces, aggregation, and formation of biofilms, is regulated by a complex mechanism. In order to identify gene(s) involved in the expression of Agf, the TnphoA transposon was introduced into Salmonella typhimurium χ8505 for random mutagenesis. Colonies showing a change from wrinkly-rough morphology to the smooth form were screened for candidates. Through multiple selection processes, a mutant, named S. typhimurium CK167 was selected as the final candidate. Analyses of the nucleotide sequences of TnphoA insertion site identified the insertion in rpoE gene. S. typhimurium CK178, a defined rpoE deletion mutant on χ8505, exhibited the same colony morphology as seen in CK167. The S. typhimurium CK178 strain expressed significantly reduced amounts of AgfD and showed modulated biofilm formation, demonstrating the role of RpoE in AgfD expression. To the best of our knowledge, this is the first report demonstrating that RpoE acts as a regulator in the expression of Agf in Salmonella.

Identification of antigenic Edwardsiella tarda surface proteins and their role in pathogenesis.

Edwardsiella tarda causes an infectious fish disease called edwardsiellosis. Several outer membrane proteins (OMPs) are associated with virulence factors and are attractive as vaccine candidates. In this study, 4 immuno-reactive OMPs of E. tarda were detected using anti-sera from flounder infected with E. tarda. Using matrix-assisted laser desorption/ionization mass spectrometry analyses, 2 of the 4 OMPs were identified as OmpA and murein lipoprotein (Lpp), which are highly conserved surface proteins in gram-negative bacteria. For further characterization of these surface proteins, we generated ompA- and lpp-inactivated mutants by insertion of a kanamycin cassette in the corresponding genes, and named these mutants E. tarda CK99 and CK164, respectively. As expected, immuno-reactive OmpA and Lpp proteins were absent in E. tarda CK99 and CK164, respectively, confirming that OmpA and Lpp are antigenic surface proteins. Interestingly, the LD(50) value of E. tarda CK164 in fish (2.0 × 10(8) colony-forming unit [CFU]/fish) was greater than that of the parental strain (3.0 × 10(7) CFU/fish). The LD(50) of E. tarda CK99 did not differ from that of its parental strain. After administering attenuated E. tarda CK164 to fish, we monitored the E. tarda-specific immune response profile. We observed that the E. tarda-specific serum IgM titer increased in a time-dependent manner, and was much higher than the value observed after the administration of a heat-killed E. tarda control. Moreover, fish vaccinated with E. tarda CK164 were 100% protected when challenged by CK41, a pathogenic strain. Our results suggest that E. tarda CK164 can potentially be used for developing an effective live attenuated vaccine for edwardsiellosis that can be applied in the aquaculture industry.

Large antibiotic-resistance plasmid of Edwardsiella tarda contributes to virulence in fish.

Edwardsiella tarda, an enteric gram negative bacterium, infects a wide range of fish and causes a systemic fish disease called edwardsiellosis. E. tarda CK41, isolated from Japanese flounder diagnosed with edwardsiellosis, has exhibited a high degree of resistance to multiple antibiotics, including kanamycin, tetracycline, streptomycin, among others. As the bacterial antibiotic-resistance genes are usually contained in plasmids, we hypothesized that E. tarda CK41 may harbor one or more plasmids for antibiotic resistance. We showed the existence of plasmids in E. tarda CK41, and the size of the plasmid, designated as pCK41, was estimated to be approximately 70 kb. Escherichia coli DH5α transformed by the pCK41 plasmid exhibited an antibiotic-resistance phenotype against kanamycin (30 µg/mL), tetracycline (30 µg/mL), and streptomycin (10 µg/mL), indicating the existence of at least 3 antibiotic-resistance genes in pCK41. Through a procedure for pCK41 plasmid curing, a plasmid-cured strain, designated as E. tarda CK108, was identified, which was unable to grow in the presence of either kanamycin or tetracycline. As virulence-associated genes are occasionally encoded in bacterial plasmids, we examined the virulence of E. tarda CK108 in Japanese flounder. The virulence of plasmid-cured E. tarda CK108 was lower (survival rate 80%) than that of CK41 (20%), indicating the existence of virulence-associated genes in pCK41. The strain also appeared to be attenuated in both goldfish and zebrafish pathogenesis models. To analyze genes for antibiotic resistance and virulence in pCK41, the entire nucleotide sequences of pCK41 were determined (GenBank accession number: HQ332785). A total of 84 open reading frames (ORFs) were annotated. The pCK41 plasmid consists of potential virulence genes, transposases, plasmid maintenance genes, antibiotic-resistance genes (including kanamycin, tetracycline, and streptomycin), conjugal transfer genes, and unknown ORFs. These results suggest that pCK41 is a virulence plasmid of substantial importance in the E. tarda pathogenesis to fish.

Requirement of Fur for the full induction of Dps expression in Salmonella enterica serovar typhimurium.

The Dps protein, which is overexpressed in harsh environments, is known to play a critical role in the protection of DNA against oxidative stresses. In this study, the roles of Fur in the expression of the dps gene in Salmonella and the protection mechanisms against oxidative stress in Salmonella cells preexposed to iron-stress were investigated. Two putative Fur boxes were predicted within the promoter region o f th e S. typhimurium dps gene . The profile of dps expression performed by the LacZ reporter assay revealed growth-phase dependency regardless of iron-status under the culture conditions. Thefur mutant, chi4659, evidenced a reduced level of beta-galactosidase as compared to the wild-type strain. The results observed after the measurement of the Dps protein in various Salmonella regulatory mutants were consistent with the results acquired in the reporter assay. This evidence suggested that Fur performs a function as a subsidiary regulator in the expression of dps. The survival ability of Salmonella strains after exposure to oxidative stress demonstrated that the Dps protein performs a pivotal function in the survival of stationary-phase S. typhimurium against oxidative stress. Salmonella cells grown in iron-restricted condition required Dps for full protection against oxidative stress. The CK24 (Deltadps) cells grown in iron-replete condition survived at a rate similar to that observed in the wild-type strain, thereby suggesting the induction of an unknown protection mechanism(s) other than Dps in this condition.

Development of a protein secretion system with the application of sec-dependent protein secretion components.

In order to induce high levels of protein secretion, we have constructed a recombinant plasmid, designated pBP244, into which was incorporated key components of the type-II Sec-dependent secretion system, including LepB (signal peptidase), SecA (ATPase), and SecB (chaperone). The biological activities of the LepB, SecA, and SecB components expressed from genes harbored by pBP244 appeared to play their normal roles. In order to evaluate the protein secretion, a pspA (Streptococcus pneumoniae surface protein A) gene was cloned into pBP244, resulting in pBP438. S. typhimurium harboring pBP438 grown until the stationary phase, secreted a higher level of PspA into the culture supernatants than did the strain harboring pYA3494. The strain harboring pBP438 secreted a supernatant amount 1.71-fold, a periplasmic space amount 1.47-fold, and an outer membrane amount 1.49-fold higher than that of pYA3494. S. typhimurium chi8554 kept the Asd+ plasmid pBP244 and pBP438 for 60 generations in LB broth harboring DAP, thereby indicating that pBP244 and pBP438 were quite stable in the Salmonella strain.

Goldfish, Carassius auratus, as an infection model for studying the pathogenesis of Edwardsiella piscicida.

This study demonstrates the feasibility of using goldfish as an infection model to investigate the pathogenesis of Edwardsiella piscicida. Goldfish were found to be susceptible to acute E. piscicida-induced disease and died in a dose-dependent manner. E. piscicida was further shown to replicate rapidly in the head kidneys and livers of infected goldfish from 1 d post-injection, and bacteria numbers were significantly decreased 5 d post-injection. Immune responses were successfully induced in goldfish injected with E. piscicida strains and 60% of goldfish inoculated with an attenuated E. piscicida strain were found to survive subsequent injection with a pathogenic strain. The results of differential leukocyte count experiments suggested that leukocytes were immediately recruited as an innate immune response against the infection. Thus, this well-characterized goldfish species is a suitable infection model for studying E. piscicida pathogenesis, and might be applicable to research on other fish diseases.

Investigation of variable lymphocyte receptors in the alternative adaptive immune response of hagfish.

Jawless vertebrates have an alternative adaptive immune system mediated by variable lymphocyte receptors (VLRs), VLRA, VLRC and VLRB. In investigation on the adaptive immunity of hagfish, avian influenza virus hemagglutinin (H9N2-HA1) was used as a model antigen, with mRNA expression levels of VLRA, VLRC and Ikaros were up-regulated in the first week post-immunization. CD45 was up-regulated after the first week; and expression of VLRB progressively increased over the course of the trial. The transcriptional/translational activation of VLRB in blood was verified. The VLRBs cloned from these transcripts showed diversity in their leucine-rich repeats (LRRs). The production of specific VLRB increased in a time- and dose-dependent manner, detected by an anti-VLRB antibody (11G5). The plasma VLRB could distinguish H9N2-HA1 from unrelated proteins, but not from other HA1 subtypes. Together, our findings show that VLRs play a major role in the alternative adaptive immune system of hagfish by responding to specific foreign substances, such as H9N2-HA1.

Development of a multiplex PCR assay to detect Edwardsiella tarda, Streptococcus parauberis, and Streptococcus iniae in olive flounder (Paralichthys olivaceus).

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.