Edwardsiella tarda DnaK: expression, activity, and the basis for the construction of a bivalent live vaccine against E. tarda and Streptococcus iniae

Edwardsiella tarda and Streptococcus iniae are important aquaculture pathogens that affect many species of farmed fish. In this study, we analyzed the expression, activity, and immunoprotective potential of E. tarda heat shock protein DnaK. We found that dnaK expression was upregulated under conditions of heat shock, oxidative stress, and infection of host cells. Recombinant DnaK (rDnaK) purified from Escherichia coli exhibited ATPase activity and induced protection in Japanese flounder (Paralichthys olivaceus) against lethal E. tarda challenge. On the basis of these results and our previous observation that a protective S. iniae antigen Sia10 which, when expressed heterogeneously in E. coli DH5α, is secreted into the extracellular milieu, we constructed a chimeric antigen by fusing DnaK to Sia10. The resulting fusion protein Sia10-DnaK was expressed in DH5α via the plasmid pTDK. Western blot analysis indicated that Sia10-DnaK was detected in the culture supernatant of DH5α/pTDK. When flounder were vaccinated with live DH5α/pTDK, strong protection was observed against both E. tarda and S. iniae. ELISA analysis detected specific serum antibody production in fish vaccinated with rDnaK and DH5α/pTDK. Taken together, these results indicate that rDnaK is an intrinsic ATPase with immunoprotective property and that Sia10-DnaK delivered by a live bacterial host is an effective bivalent vaccine candidate against E. tarda and S. iniae infection.     

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 LD₅₀ value of E. tarda CK164 in fish (2.0 × 10⁸ colony-forming unit [CFU]/fish) was greater than that of the parental strain (3.0 × 10⁷ CFU/fish). The LD₅₀ 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.     

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

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.     

Invasin of Edwardsiella tarda is essential for its haemolytic activity,biofilm formation and virulence towards fish

Aims

The aim of this study was to investigate the role of invasin in a bacterial fish pathogen Edwardsiella tarda.

Methods and Results

In this study, an in‐frame deletion mutant of invasin (Δinv) in Edw. tarda H1 was constructed through double crossover allelic exchange to explore the function of invasin in virulence to fish. Meanwhile, an invasin overexpression strain (inv⁺) was obtained by electrotransformation of a low‐copy plasmid pACYC184 carrying the intact invasin into the Δinv mutant. Several virulence‐associated characters of the mutants and wild‐type strain were tested. Compared with the wild‐type H1, haemolytic activity and biofilm formation were decreased in Δinv, while increased significantly in inv⁺. In addition, the invasin overexpressing strain inv⁺ exhibited increased internalization into Epithelioma Papulosum Cyprini (EPC) cells. Moreover, in zebrafish model, Δinv showed decreased virulence compared with H1, while inv⁺ restored the virulence of wild type completely.

Conclusions

The results demonstrated that invasin of Edw. tarda plays essential roles in haemolytic activity, biofilm formation, adherence, internalization and pathogenicity of this bacterium.

Significance and Impact of the Study

This study revealed the role of invasin in Edw. tarda infection and provided useful information for further unveiling the pathogenesis of Edw. tarda.     

First molecular cloning and gene expression analysis of a teleost CD200 (OX-2) glycoprotein from rock bream,Oplegnathus fasciatus

CD200 plays an important role in delivering an immunoregulatory signal to the immune system through interaction with its receptor. However, CD200 has not been characterized and its function in teleosts is unknown. In this study, the rock bream (Oplegnathus fasciatus) CD200 gene (RbCD200) was cloned and its expression profile was analyzed after infection with Edwardsiella tarda, Streptococcus iniae or red seabream iridovirus (RSIV). The coding region of RbCD200 cDNA was 855 bp, encoding 284 amino acid residues. The gene consisted of two extracellular Ig-like domains and a transmembrane domain. RbCD200 was highly expressed in the brain, erythrocytes, intestine and stomach of healthy rock bream. In the spleen, RbCD200 gene expression was down-regulated until 48 h after E. tarda exposure, except at 12 h RbCD200 gene expression was down-regulated then up-regulated at 12 h and 24 h after infection with S. iniae and RSIV, respectively. In the whole kidney, the RbCD200 gene was down-regulated in response to infection with E. tarda and S. iniae. However, RSIV infection increased RbCD200 gene expression in whole kidney until 48 h. These results suggest that RbCD200 is differentially expressed in the spleen and whole kidney after infection with different pathogens.     

A solute-binding protein for iron transport in <Emphasis Type="Italic">Streptococcus iniae</Emphasis>

Background  

Streptococcus iniae (S. iniae) is a major pathogen that causes considerable morbidity and mortality in cultured fish worldwide. The pathogen's ability to adapt to the host affects the extent of infection, hence understanding the mechanisms by which S. iniae overcomes physiological stresses during infection will help to identify potential virulence determinants of streptococcal infection. Grow S. iniae under iron-restricted conditions is one approach for identifying host-specific protein expression. Iron plays an important role in many biological processes but it has low solubility under physiological condition. Many microorganisms have been shown to be able to circumvent this nutritional limitation by forming direct contacts with iron-containing proteins through ATP-binding cassette (ABC) transporters. The ABC transporter superfamilies constitute many different systems that are widespread among living organisms with different functions, such as ligands translocation, mRNA translation, and DNA repair.     

Evolution of the Capsular Operon of Streptococcus iniae in Response to Vaccination

Streptococcus iniae causes severe septicemia and meningitis in farmed fish and is also occasionally zoonotic. Vaccination against S. iniae is problematic, with frequent breakdown of protection in vaccinated fish. The major protective antigens in S. iniae are the polysaccharides of the capsule, which are essential for virulence. Capsular biosynthesis is driven and regulated by a 21-kb operon comprising up to 20 genes. In a long-term study, we have sequenced the capsular operon of strains that have been used in autogenous vaccines across Australia and compared it with the capsular operon sequences of strains subsequently isolated from infected vaccinated fish. Intriguingly, strains isolated from vaccinated fish that subsequently become infected have coding mutations that are confined to a limited number of genes in the cps operon, with the remainder of the genes in the operon remaining stable. Mutations in strains in diseased vaccinated fish occur in key genes in the capsular operon that are associated with polysaccharide configuration (cpsG) and with regulation of biosynthesis (cpsD and cpsE). This, along with high ratios of nonsynonymous to synonymous mutations within the cps genes, suggests that immune response directed predominantly against capsular polysaccharide may be driving evolution in a very specific set of genes in the operon. From these data, it may be possible to design a simple polyvalent vaccine with a greater operational life span than the current monovalent killed bacterins.     

Vaccination of sex reversed hybrid tilapia (Oreochromis niloticus × O. aureus) with an inactivated Vibrio vulnificus vaccine

Vibrio vulnificus causes disease in economically important aquaculture raised fish and is an opportunistic human pathogen. This study reports on the isolation of V. vulnificus from diseased hybrid tilapia (Oreochromis niloticus × O. aureus) cultured in a North American water reuse facility. Our objectives were to characterize the isolate using biochemical and molecular methods, develop a disease challenge model, and determine the ability of a formalin inactivated whole-cell vaccine to protect against V. vulnificus. The V. vulnificus isolate recovered was biotype 1, 16S rRNA type B, vcg type C, and vvhA type 2 and caused disease in tilapia held in static salt water (1.5 g/l sea salt). Fish vaccinated with the formalin inactivated whole-cell vaccine responded to vaccination with titers from vaccinated fish ranging from 32 to 64 and titers from non-vaccinated fish ranging from 4 to 8. In two trials, vaccinated tilapia exhibited relative percent survival (RPS) of 73 and 60% following homologous isolate challenge. In two additional trials, vaccinated tilapia exhibited RPS values of up to 88% following challenge with a heterologous isolate; the use of a mineral oil adjuvant enhanced protection. This vaccine may provide an effective means of preventing infections caused by biochemically and genetically diverse V. vulnificus.     

Molecular cloning and characterization of Toll-like receptor 14 in Japanese flounder,Paralichthys olivaceus

Toll-like receptors (TLRs) are essential for activation of the innate immune system in response to invading pathogens. TLR14, which is unique to fish, has been identified in several fish species, but its function is unclear. In this study, Japanese flounder (Paralichthys olivaceus) TLR14 gene (JfTLR14) was cloned and its expression profiles were analyzed after infection with viral hemorrhagic septicemia virus, gram-positive Streptococcus iniae and gram-negative Edwardsiella tarda. The coding region of JfTLR14 cDNA was 2,607 bp, encoding 878 amino acid residues. JfTLR14 was highly expressed in head kidney of healthy flounder. In response to infection with VHSV and S. iniae, the JfTLR14 gene was up-regulated at only 1 day post-infection (dpi). However, E. tarda infection increased JfTLR14 gene expression from 1 to 6 dpi. These results imply that JfTLR14 participates more in the immune response against E. tarda infection than in the immune responses to other pathogen infections.     

Asd-based balanced-lethal system in attenuated Edwardsiella tarda to express a heterologous antigen for a multivalent bacterial vaccine

Edwardsiella tarda is an enteric Gram-negative invasive intracellular pathogen, which causes enteric septicemia in fish. It could be potentially used to develop a recombinant attenuated E. tarda vaccine for the aquaculture industry. Because live vaccine strains can potentially be released into the environment upon vaccination, medical and environmental safety issues must be considered. Deletion of the asdB gene in E. tarda resulted in a diaminopimelic acid (DAP)-dependent mutant. The wild type asdB gene was inserted in place of the antibiotic-resistance gene in the plasmid, and the resultant non-antibiotic resistant vector was transformed into the attenuated and DAP-dependent E. tarda vaccine strain (WEDΔasdB) to obtain a balanced-lethal system for heterologous antigen expression. The balanced-lethal expression system was further optimized by comparing plasmid replicons with different Shine–Dalgarno sequences and start codons for the asdB gene. Utilizing the optimized balanced-lethal expression system, the protective antigen gene gapA34 from the fish pathogen Aeromonas hydrophila LSA34 was expressed in the attenuated E. tarda to generate the multivalent vaccine candidate WEDΔasdB/pUTta4DGap. This vaccine was shown to evoke an effective immune response against both E. tarda and A. hydrophila LSA34 by vaccinating turbot via a simple immersion route. This multivalent E. tarda vector vaccine has great potential for broad applications in aquaculture.     

Growth and thermal tolerance of a Tibet fish Schizopygopsis younghusbandi juveniles acclimated to three temperature levels

Schizopygopsis younghusbandi is an endemic fish of Tibet characterized by slow growth. Artificial stock enhancement was applied to rebuild the natural population of S. younghusbandi in recent years. However, the optimal growth temperature and thermal tolerance of S. younghusbandi has not been studied, which restricts the production of S. younghusbandi fingerling for stock enhancement. The purpose of this paper is to determine the growth, critical thermal maximum (CTMax), lethal thermal maximum (LTMax) and acclimation response ratio (ARR) of S. younghusbandi juveniles (body weight 5.7 ± 1.2 g) at three acclimation temperature levels (10, 15, 20°C). The results showed that acclimation temperature significantly affected the growth, CTMax, LTMax and ARR of the experimental fish. Largest final weight (7.5 ± 2.3 g) was recorded in 15°C group. At a heating rate of 1°C/30 min, CTMax ranged from 30.98 to 32.01°C and LTMax ranged from 31.76 to 32.31°C in the three acclimation temperatures. Schizopygopsis younghusbandi had lower ARR value (0.097) than most other fish species. Low ARR value indicates that S. younghusbandi may have narrower thermal tolerance range and weaker acclimation ability to global warming. For successful aquaculture of S. younghusbandi juveniles, temperature should be maintained around 15°C.     

Analysis of Edwardsiella tarda DegP,a serine protease and a protective immunogen

Edwardsiella tarda is a severe aquaculture pathogen with a broad host range that includes humans, animal, and fish. A gene (degP_Et) encoding a DegP homologue was cloned from TX01, a pathogenic E. tarda strain isolated from diseased fish. DegP_Et shares high sequence identities with the DegP proteins of several bacterial species. Functional analyses showed that degP_Et could complement the temperature-sensitive phenotype of an Escherichia coli degP null mutant. Expression of degP_Et in TX01 was modulated by growth phase and temperature, the latter possibly through the action of the σ^E-like factor. Overexpression of degP_Et (i) enhanced the ability of TX01 to disseminate in fish blood at the advanced stage of infection, (ii) heightened the activity of type 2 autoinducer, and (iii) increased the expression of luxS and the genes encoding components of the virulence-associated type III secretion system. Recombinant DegP_Et purified from E. coli was a serine protease that exhibited maximum activity at 40 °C and pH8.0. The proteolytic activity of recombinant DegP_Et depended on the catalytic triad and the PDZ domains. Immunoprotective analyses showed that purified recombinant DegP_Et was a protective immunogen that could induce the production of specific serum antibodies and elicit strong protective immunity in fish vaccinated with DegP_Et.     

Tissue expression and bioinformatics analysis of the vitellogenin gene of Asian arowana (Scleropages formosus)

The RACE technique was used to clone the full‐length vitellogenin (VTG) cDNA sequence of Asian arowana (Scleropages formosus). The full‐length sequence was 5,550 bp with an open reading frame of 5,238 bp, encoding 1,745 amino acids, and 5′ and 3′ UTRs (untranslated regions) of 45 bp and 267 bp, respectively. Phylogenetic analysis showed that S. formosus and silver arowana (Osteoglossum bicirrhosum) share a close evolutionary relationship (bootstrap 100%). The quantitative real‐time PCR results showed that vtg expression was significantly higher in liver and gonads of male and female fish compared with its expression in the other tissues tested (p < 0.01). The relative expression levels of vtg in liver, gland, kidney, heart, head kidney, and brain of female fish were significantly higher than in the corresponding tissues of male fish (p < 0.05).     

Salt effect of nisin Z isolated from a marine fish on the growth inhibition of Streptococcus iniae, a pathogen of streptococcosis

A bacteriocin-producing Lactococcus lactis subsp. lactis was isolated from the intestine of olive flounder. The bacteriocin was identified as nisin Z. It was active against Gram-positive bacteria. Nisin Z at 3,200 arbitrary units (AU) was more effective in seawater than in PBS; growth of Streptococcus iniae was completely inhibited within 3 h. Nisin Z preparations with 3.5% (w/v) NaCl was the most effective against S. iniae being similar to nisin Z in seawater. Nisin Z is thus a good alternative to antibiotics to prevent streptococcosis caused by S. iniae aquaculture systems.     

Comparative morphology of sensilla on antenna,maxillary palp and labial palp of larvae of Eucryptorrhynchus scrobiculatus (Olivier) and E. brandti (Harold) (Coleoptera: Curculionidae)

Eucryptorrhynchus scrobiculatus (Olivier) and E. brandti (Harold) are two wood boring pests of Ailanthus altissima (Mill.) Swingle (tree of heaven) and the variety Ailanthus altissima var. Qiantouchun. These beetles attack healthy trees and bore into roots and trunks during the larval stage. We studied the typology, distribution and morphostructure of the sensilla on the antennae, maxillary palps and labial palps of E. scrobiculatus and E. brandti larvae using scanning and transmission electron microscopy. The results showed the following: (i) the antennae of the two weevil larvae had two types of sensilla, sensilla basiconica (S.b.1 and S.b.2) and sensilla twig basiconica (S.tb.1‐S.tb.3), with S.tb.4 observed only on the antennae of E. brandti larvae; (ii) the maxillary palps had three types of sensilla, S.b.2, S.tb. (S.tb.2, S.tb.3 and S.tb.5) and digitiform sensilla; (iii) the labial palps had two types of sensilla, S.b.2 and S.tb. (S.tb.2, S.tb.3 and S.tb.5); (iv) the quantity and distribution of sensilla on the antennae, maxillary palps and labial palps remained constant between E. scrobiculatus and E. brandti larvae; and (v) sensilla basiconica had distinct sidewall pores, an apical pore was observed on sensilla twig basiconica, and digitiform sensilla were oval in shape, with a distinct apical pore. Based on the microstructure of the cuticle wall and dendrite, we hypothesized that these sensilla functioned as olfactory, gustatory and hygro‐/thermo‐receptors, respectively. We discuss the relationships among types of sensilla and the types of damage caused by the larvae inside the host tree to understand olfactory and gustatory receptor mechanisms. The results of this study will provide a firm basis for future electrophysiological studies.     

The influence of prey size,sediment thickness and fish size on consumption in common sole (Solea solea L.)

This study determined prey consumption in common sole as a function of prey size (0–0.5, 1–1.5, 2–2.5 and 4–5 g), sediment thickness (20 cm and 2 cm) and fish size (50 g, 125 g or 300 g). Prey consumption (in numbers of prey eaten per fish per day) was reduced with increasing prey size and sediment thickness, and was increased with increasing fish size (p < .001 for all factors). All 3 factors showed significant two way interactions (p < .001) when expressed in numbers of prey eaten. Prey consumption decreased with prey size when prey could not escape by burying (2 cm of sediment thickness) irrespective of fish size. We suggest that increasing effort to ingest and handle larger prey played a role. Prey consumption increased with fish size when prey could not bury (2 cm of sediment thickness). However, when prey was able to bury (at 20 cm sediment thickness) prey consumption was similar irrespective of fish size (p < .001 for interaction fish size × sediment). This interaction suggests that with increasing fish size there is an increasing mismatch between foraging adaptation and prey burial depth. This may explain the dominance of crustaceans in the diet of adult common sole in nature, despite the high abundance of polychaetes.