Mobilisation and dysfunction of haematopoietic stem/progenitor cells after Listonella anguillarum infection in ayu, Plecoglossus altivelis.

Haematopoietic stem/progenitor cells (HSPCs) can mobilise into blood and produce immune cell lineages following stress. However, the homeostasis and function of HSPCs after infection in teleosts are less well known. Here, we report that Listonella anguillarum infection enhances HSPC mobilisation and reduces their differentiation into myeloid cells in ayu (Plecoglossus altivelis), an aquacultured teleost in East Asia. We established a colony-forming unit culture (CFU-C) assay to measure HSPCs using conditioned medium from peripheral blood mononuclear cells stimulated with phytohaemagglutinin. The number of CFU-Cs decreased in the head kidney and increased in the blood and spleen of ayu infected with L. anguillarum. HSPC mobilisation after L. anguillarum infection was mediated by norepinephrine. Furthermore, HSPCs from ayu treated with L. anguillarum lipopolysaccharide (LPS) showed defective myeloid differentiation and could no longer rescue L. anguillarum-infected ayu. HSPC expansion was suppressed after L. anguillarum infection or its LPS treatment in vitro. These results reveal a link between HSPC regulation and pathogen infection in teleosts.

Succinate dehydrogenase mutant of Listonella anguillarum protects rainbow trout against vibriosis.

Listonella anguillarum is a Gram-negative facultative anaerobic rod causing hemorrhagic septicemia in marine and rarely in freshwater fish. Succinate dehydrogenase (SDH) plays an important role in the tricarboxylic acid (TCA) cycle by oxidizing succinate to fumarate while reducing ubiquinone to ubiquinol. Recent studies indicate that central metabolic pathways, including the TCA cycle, contribute to bacterial virulence. However, the role of SDH in L. anguillarum virulence has not been studied. Here, we report in-frame deletion of the succinate dehydrogenase iron-sulfur protein (SDHB) and its role in L. anguillarum virulence in rainbow trout. To accomplish this goal, upstream and downstream regions of the L. anguillarum sdhB gene were amplified in-frame and cloned into a suicide plasmid. The chromosomal sdhB gene of L. anguillarum was deleted by homologous recombination. Virulence and immunogenicity of the L. anguillarum ΔsdhB mutant (LaΔsdhB) were determined in rainbow trout. Results show that LaΔsdhB was highly attenuated in rainbow trout, and fish immunized with LaΔsdhB displayed high relative survival rate after exposure to wild type L. anguillarum. These findings indicate SDH is important in L. anguillarum virulence in rainbow trout, and LaΔsdhB could be used as an immersion, oral, or injection vaccine to protect rainbow trout against vibriosis.

Pathogenicity of members of the vibrionaceae family to cultured juvenile sablefish.

Sablefish Anoplopoma fimbria are a prized seafood species due to their high oil content and white flaky flesh. Raising these species in culture can help to provide an important source of protein for humans and relief to declining wild fish populations. Understanding the environmental factors that influence the production of Sablefish is important for successful culturing. The significance of host-pathogen interactions in Sablefish culture and the resulting environmental implications are unknown. Pathogens could potentially cause losses of cultured Sablefish stocks due to disease, while Sablefish cultured in net pens may also serve as reservoirs for pathogens and potentially transmit disease to wild fish species. In this initial study, the susceptibility of juvenile Sablefish to three bacterial pathogens from the family Vibrionaceae was examined. Listonella anguillarum, Vibrio ordalii, and V. splendidus can pose serious economic threats to cultured fish and shellfish. Groups of juvenile Sablefish were exposed to five concentrations of each of the pathogens. Sablefish were susceptible to L. anguillarum, but were resistant to V. ordalii and V. splendidus at exposure concentrations of ≤ 1.32 × 107 CFU/mL and ≤ 3.57 × 106 CFU/mL, respectively. The greatest L. anguillarum concentration examined (8.7 × 106 CFU/mL) resulted in 24% mortality in juvenile Sablefish. A 24% loss of Sablefish stock could significantly influence an aquaculture program. As determined by multiple logistic regression, the survival of Sablefish to L. anguillarum exposure was significantly affected by their body mass, and larger fish had a greater probability of survival. Aquaculture operations could employ various strategies to minimize the loss of juvenile Sablefish by accounting for their size and known susceptibilities to pathogens.

Molecular characterization of a CXCL8-like protein from ayu and its effect on chemotaxis of neutrophils and monocytes/macrophages.

CXCL8, a CXC-type chemokine, plays a crucial role in acute inflammation by recruiting and mediating neutrophils and other cells. In this study, the cDNA and genomic DNA sequence of a CXCL8-like protein (PaCXCL8l) from ayu (Plecoglossus altivelis) was determined. Sequence analysis showed that PaCXCL8l represented the typical structure of animal CXCL8s. Phylogenetic tree analysis indicated that PaCXCL8l was closest to CXCL8 of Atlantic cod (Gadus morhua). Constitutive expression of PaCXCL8l was detected in all tested tissues and monocytes/macrophages, and its expression dramatically increased upon Listonella anguillarum infection. In vitro, recombinant PaCXCL8l exhibited a significant chemotactic effect on neutrophils at 0.1 µg/ml and on monocytes/macrophages at 1.0 µg/ml. In vivo, the numbers of peritoneal neutrophils and monocytes/macrophages were both up-regulated following intraperitoneal administration of recombinant PaCXCL8l. These results suggest that PaCXCL8l is crucially involved in the immune response of ayu by mediating chemotaxis of neutrophils and monocytes/macrophages.

A γ-aminobutyrate type A receptor-associated protein involved in the immune response of Eriocheir sinensis.

The γ-aminobutyrate type A receptor-associated protein (GABARAP) is a ubiquitin-like modifier, which is implicated in membrane trafficking and fusion events of γ-aminobutyrate type A receptor, autophagy and apoptosis. In the present study, the gene encoding GABARAP (designated EsGABARAP) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends approach and expression sequence tag (EST) analysis. The full-length cDNA of EsGABARAP was of 457 bp, containing a 5' untranslated region (UTR) of 77 bp, a 3' UTR of 32 bp with a poly(A) tail and an open reading frame (ORF) of 348 bp encoding a polypeptide of 116 amino acids with the predicted molecular weight of 13.81 kDa and theoretical isoelectric point of 8.73. The deduced amino acid sequence of EsGABARAP shared higher similarity (91.8-97.4%) with those of other GABARAPs, and it contained a conserved MAP1_LC3 domain. In the phylogenetic tree, EsGABARAP was firstly clustered with GABARAPs from other animals and then gathered together with the same family proteins of GABARAP. The mRNA expression level of EsGABARAP in six tissues and its temporal expression level in haemocytes of crabs challenged with Listonella anguillarum were determined by quantitative real-time RT-PCR. The mRNA transcripts of EsGABARAP could be detected ubiquitously in the examined tissues, including haemocytes, hepatopancreas, muscle, gill, heart and gonad, with the highest expression level in hepatopancreas. The expression level of EsGABARAP mRNA in haemocytes was up-regulated after L. anguillarum challenge and reached 6.58-fold of that in blank group at 24 h (P < 0.05) and 7.52-fold at 48 h (P < 0.05). The increasing transcripts in haemocytes after L. anguillarum challenge gave the preliminary evidence for the involvement of EsGABARAP as a part of immune response against bacteria challenge in crabs.

The second anti-lipopolysaccharide factor (EsALF-2) with antimicrobial activity from Eriocheir sinensis.

The anti-lipopolysaccharide factor (ALF) is a small basic protein that can bind and neutralize lipopolysaccharide (LPS), mediating degranulation and activation of an intracellular coagulation cascade. In the present study, cDNA of the second Eriocheir sinensis ALF (designated as EsALF-2) was cloned and the full-length cDNA of EsALF-2 was of 724bp, consisting of an open reading frame (ORF) of 363bp encoding a polypeptide of 120 amino acids. The deduced amino acid of EsALF-2 shared 82% similarity with EsALF-1 from E. sinensis and about 53-65% similarity with ALFs from other crustaceans. The potential tertiary structures of EsALF-1 and EsALF-2 contained two highly conserved-cysteine residues to define the LPS binding site, but the N-terminal of EsALF-1 formed a single additional alpha-helix compared to EsALF-2, implying that EsALF-1 and EsALF-2 might represent different biological functions in E. sinensis. The mRNA transcript of EsALF-2 was detected in all examined tissues of healthy crabs, including haemocytes, hepatopancreas, gill, muscle, heart and gonad, which suggested that EsALF-2 could be a multifunctional molecule for the host immune defense responses and thereby provided systemic protection against pathogens. The mRNA expression of EsALF-2 was up-regulated after Listonella anguillarum and Pichia pastoris challenge and the recombinant protein of EsALF-2 showed antimicrobial activity against L. anguillarum and P. pastoris, indicating that EsALF-2 was involved in the immune defense responses in Chinese mitten crab against L. anguillarum and P. pastoris. These results together indicated that there were abundant and diverse ALFs in E. sinensis with various biological functions and these ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control of crab aquaculture.

Characterization of chitinolytic bacteria and histological aspects of Shell Disease Syndrome in European spiny lobsters (Palinurus elephas) (Fabricius 1787).

The present research reports the first description of Shell Disease Syndrome in European spiny lobsters Palinurus elephas (Fabricius 1787), which occurred in an experimental aquaculture facility in Sicily (Italy). Both bacterial characterization and histopathological examination of the exoskeleton at site of lesions was carried out. Infected specimens showed tail fan erosions, and in one case uropod ulceration and complete loss of periods. Identified species included: Listonella anguillarum 50.5%, Vibrio parahaemolyticus 27.5% and Vibrio alginolyticus 22%. Microscopic evaluation of lesions indicate the presence of inflammatory responses, which include melanization and pseudomembrane formation, similar to those described for other crustaceans affected by SDS.

AiC1qDC-1, a novel gC1q-domain-containing protein from bay scallop Argopecten irradians with fungi agglutinating activity.

The globular C1q-domain-containing (C1qDC) proteins are a family of versatile pattern recognition receptors via their globular C1q (gC1q) domain to bind various ligands including several PAMPs on pathogens. In this study, a new gC1q-domain-containing protein (AiC1qDC-1) gene was cloned from Argopecten irradians by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNA of AiC1qDC-1 was composed of 733bp, encoding a signal peptide of 19 residues and a typical gC1q domain of 137 residues containing all eight invariant amino acids in human C1qDC proteins and seven aromatic residues essential for effective packing of the hydrophobic core of AiC1qDC-1. The gC1q domain of AiC1qDC-1, which possessed the typical 10-stranded beta-sandwich fold with a jelly-roll topology common to all C1q family members, showed high homology not only to those of C1qDC proteins in mollusk but also to those of C1qDC proteins in human. The AiC1qDC-1 transcripts were mainly detected in the tissue of hepatopancreas and also marginally detectable in adductor, heart, mantle, gill and hemocytes by fluorescent quantitative real-time PCR. In the microbial challenge experiment, there was a significant up-regulation in the relative expression level of AiC1qDC-1 in hepatopancreas and hemocytes of the scallops challenged by fungi Pichia pastoris GS115, Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Listonella anguillarum. The recombinant AiC1qDC-1 (rAiC1qDC-1) protein displayed no obvious agglutination against M. luteus and L. anguillarum, but it aggregated P. pastoris remarkably. This agglutination could be inhibited by d-mannose and PGN but not by LPS, glucan or d-galactose. These results indicated that AiC1qDC-1 functioned as a pattern recognition receptor in the immune defense of scallops against pathogens and provided clues for illuminating the evolution of the complement classical pathway.

Expression of antimicrobial peptides from Hyas araneus haemocytes following bacterial challenge in vitro.

Circulating haemocytes play major roles in the host defense reactions of decapods, including the synthesis and release of antimicrobial peptides (AMPs). Unlike the AMPs from insects, those in decapods are constitutively expressed. This study aims to establish primary cell cultures of the three main haemocyte types in Hyas araneus haemocytes, and to measure the in vitro expression of AMP genes in the cells following microbial challenge. The haemocyte populations were separated on Percoll gradients and cultured in modified L-15 medium. Expression analysis by real-time RT-PCR showed that the granular cells are the main producers of crustin, hyastatin and arasin 1 AMPs, but the hyaline cells and semigranular cells also show some expression of these genes. Incubating the cell populations with Aerococcus viridans var. homari (a Gram-positive bacterium) or Listonella anguillarum (a Gram-negative pathogen) provoked no dramatic changes in the gene expression of any of the AMP, and although there was a small (single doubling) significant increase in expression of the crustin gene in granular cells 24h after exposure to L. anguillarum, it is unclear if this is biologically relevant under in vitro conditions. The results presented in this study are in accordance with several in vivo studies.

Development of a bacterial challenge test for gnotobiotic sea bass (Dicentrarchus labrax) larvae.

The use of probiotic microorganisms in aquaculture is gaining a lot of interest. Gnotobiotic model systems are required in order to fully understand the effects and modes-of-action of these microorganisms, as the native microbial communities present in non-sterile animals can lead to false conclusions. In this study, a gnotobiotic sea bass larvae (Dicentrarchus labrax) test system was developed. In order to obtain bacteria-free animals, the eggs were disinfected with glutaraldehyde and subsequently incubated in a solution of rifampicin and ampicillin. Axenity was confirmed using culture-dependent and -independent techniques. The gnotobiotic larvae were fed axenic Artemia sp. from 7 days after hatching onwards. In the challenge test, one of the three opportunistic pathogens, Aeromonas hydrophila, Listonella anguillarum serovar O1 and O2a, was added to the model system via the water and encapsulated in Artemia sp. Only serovar O2a led to increased mortality in the sea bass larvae. The presented gnotobiotic model can be used for research on, among others, reciprocal metabolic effects between microorganisms and the host (e.g. as measured by gene expression), immunostimulants, pharmacological research and the histological development of the gastrointestinal tract and growth of larvae.

Alteration of metallothionein mRNA in bay scallop Argopecten irradians under cadmium exposure and bacteria challenge.

Metallothionein (MT) is a superfamily of cysteine-rich proteins contributing to metal metabolism, detoxification of heavy metals, and immune response such as protecting against ionizing radiation and antioxidant defense. A metallothionein (designated AiMT2) gene was identified and cloned from bay scallop, Argopecten irradians. The full length cDNA of AiMT2 consisted of an open reading frame (ORF) of 333 bp encoding a protein of 110 amino acids, with nine characteristic Cys-X-Cys, five Cys-X-X-Cys, five Cys-X-X-X-Cys and two Cys-Cys motif arrangements and a conserved structural pattern Cys-x-Cys-x(3)-Cys-Tyr-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Arg at the C-terminus. The cloned AiMT showed about 50% identity in the deduced amino acid sequence with previously published MT sequences of mussels and oysters. The conserved structural pattern and the close phylogenetic relationship of AiMT2 shared with MTs from other mollusc especially bivalves indicated that AiMT2 was a new member of molluscan MT family. The mRNA transcripts in hemolymph of AiMT2 under cadmium (Cd) exposure and bacteria challenge were examined by real-time RT-PCR. The mRNA expression of AiMT2 was up-regulated to 3.99-fold at 2 h after Listonella anguillarum challenge, and increased drastically to 66.12-fold and 126.96-fold at 16 and 32 h post-challenge respectively. Cadmium ion exposure could induce the expression of AiMT2, and the expression level increased 2.56-fold and 6.91-fold in hemolymph respectively after a 10-day exposure of 100 microg L(- 1) and 200 microg L(- 1) CdCl(2). The sensitivity of AiMT2 to bacteria challenge and cadmium stress indicated it was a new Cd-dependent MT in bay scallop and also regulated by an immune challenge. The changes in the expression of AiMT2 could be used as an indicator of exposure to metals in pollution monitoring programs and oxidative stress, and bay scallop as a potential sentinel organism for the cadmium contamination in aquatic environment.

The enhanced immune protection of Zhikong scallop Chlamys farreri on the secondary encounter with Listonella anguillarum.

It is well known that invertebrates are devoid of adaptive immune components and rely primarily on innate immunity to defend against pathogens, but recent studies have demonstrated the existence of enhanced secondary immune protection in some invertebrates. In the present study, the cumulative mortality of scallops received two successive Listonella anguillarum stimulations was recorded, and variations of immune parameters including phagocytosis (phagocytic rate and phagocytic index), phenoloxidase-like enzyme, acid phosphatase and superoxide dismutase activities were also examined. The scallops received a previous short-term L. anguillarum stimulation were protected against a long-term stimulation of L. anguillarum. Significantly higher level of phagocytic activities and acid phosphatase activity were observed in the scallops received twice stimulations compared with those only received the secondary stimulation. These results indicated that a short-term immersion with L. anguillarum modulated the scallops' immune system and endowed the scallops with enhanced resistance to the secondary bacterial stimulation; phagocytosis and acid phosphatase were suspected to be involved in the protection.

Decreased mortality of Lake Michigan Chinook salmon after bacterial kidney disease challenge: evidence for pathogen-driven selection?

In the late 1960s, Chinook salmon Oncorhynchus tshawytscha from the Green River, Washington, were successfully introduced into Lake Michigan. During spring from 1988 to 1992, large fish die-offs affecting Chinook salmon occurred in the lake. Multiple ecological factors probably contributed to the severity of the fish kills, but the only disease agent found regularly was Renibacterium salmoninarum, the causative agent of bacterial kidney disease. In this study, survival after challenge by R. salmoninarum was compared between two Chinook salmon stocks: a Lake Michigan stock from Wisconsin (WI) and the progenitor stock from the Green River. We found that the WI stock had significantly greater survival than the Green River stock. Next, the WI and Green River stocks were exposed to the marine pathogen Listonella anguillarum (formerly Vibrio anguillarum), one of the causative agents of vibriosis; survival after this challenge was significantly poorer for the WI stock than for the Green River stock. A close genetic relationship between the Green River and WI stocks was confirmed by analyzing 13 microsatellite loci. These results collectively suggest that disease susceptibility of Lake Michigan Chinook salmon has diverged from that of the source population, possibly in response to pathogen-driven selection.

Mannose binding lectin (MBL) copy number polymorphism in Zebrafish (D. rerio) and identification of haplotypes resistant to L. anguillarum.

We describe a novel extension of the Genomic Matching Technique (GMT) that defines haplotypes of the mannose binding lectin (MBL) region in Zebrafish (D. rerio). Four ancestral haplotypes have been identified to date, with at least one of these demonstrating a significant increase in resistance to L. anguillarum. MBL activates the lectin pathway of the complement system and stimulates the development of the complement cascade and the Membrane Attack Complex. Polymorphisms in humans have been associated with increased susceptibility and severity to a number of pathogenic organisms. As teleosts have a relatively immature acquired immune system, polymorphisms within MBL and other innate defence genes are likely to be critical in defining their susceptibility/resistance to various pathogenic organisms. We report multiple copies of MBL-like genes in D. rerio, with up to three copies tightly linked within a cluster spanning approximately 15 kb on chromosome 2. Genomic analysis suggests that duplication, retroviral insertion and possibly gene mutation and/or deletion have been key factors in the evolution of this cluster. Molecular analysis has revealed extensive polymorphism, including at least five distinct amplicons and haplospecific gene copy number variation. This study demonstrates polymorphism within a critical component of the teleost innate immune system. The polymorphisms and the haplotypes encoding the unique variants are likely to be informative in defining susceptibility/resistance to infectious agents commonly encountered within aquatic environments. Future investigations will define other important haplotypes and transfer the knowledge to other finfish species, thereby enabling selection of broodstock for the aquaculture industry.

Molecular cloning and expression analysis of a hepcidin antimicrobial peptide gene from turbot (Scophthalmus maximus).

Antimicrobial peptides (AMPs) are regarded as important components of the host innate immune system and play crucial roles in host defence against microbial invasion. A small number of hepcidin AMPs have been isolated from teleosts. Here, we report the isolation of a hepcidin gene from the liver of turbot (Scophthalmus maximus) (GenBank accession numbers: and ). In the 1037 bp-long genomic sequence, three exons and two introns were identified. The full-length cDNA is 778bp long and contains an ORF of 273bp encoding a prepropeptide of 90 amino acid residues. The predicted prepropeptide consists of three domains: a signal peptide (24 amino acids), a prodomain (40 amino acids) and a mature peptide (26 amino acids). RT-PCR demonstrated that hepcidin transcripts were highly abundant in liver, abundant in heart, head kidney, spleen, skin and gill, less abundant in blood cell, gonad and intestine, and undetectable level in muscle. The level of the hepcidin mRNA in embryos gradually increases during embryogenesis from 2 h (2 cell stage) to 95 h (larva stage) after fertilisation. Challenge of turbot with pathogenic bacteria, Listonella anguillarum, significantly elevated hepcidin mRNA levels in liver and spleen in a time-dependent fashion. The hepcidin transcripts were detected in turbot embryonic cell line (TEC). Challenge of TEC cells with the pathogenic bacteria significantly elevated hepcidin mRNA levels.