Cloning and immune characterization of the c-type lysozyme gene in red-spotted grouper,Epinephelus akaara

Lysozyme is an important component of the innate immune response against pathogen infection. The gene coding for c-type lysozyme in red-spotted grouper Epinephelus akaara was cloned and designated EaClys. The complete cDNA contains a 432 bp open reading frame encoding a protein of 144 amino acids displaying 65–91% similarity with the amino acid sequences of human, mouse, chicken, and fish counterparts. Recombinant EaClys (rEaClys) was expressed in Escherichia coli, displayed antibacterial activity against Gram-positive and Gram-negative bacteria, and possessed bactericidal activity against Vibrio alginolyticus. EaClys mRNA was constitutively expressed in all tested E. akaara tissues, and its expression increased after pathogen challenge. Most notably, challenges with LPS, SGIV or V. alginolyticus upregulated EaClys mRNA expression in the head, kidney, and blood. Its expression peaked between 16 and 24 h after challenge before dropping back to the baseline level. By using recombinant cytokines as signaling pathway mimetics and blocking antibodies and chemical inhibitors as pathway inhibitors, we show that LPS-induced lysozyme release from macrophages is promoted by cytokines TNF-α and IL-1β, and dependent on NF-κB pathway activation. These data suggest that EaClys is a constitutive and inducible acute-phase protein that is involved in the innate immune defense of E. akaara, and provide new clues about the molecular mechanisms that regulate innate immune responses in fish.     

Enzymatic,outer membrane proteins and plasmid alterations of starved <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> cells in seawater

The marine bacteria Vibrio parahaemolyticus and V. alginolyticus were incubated in seawater for 8 months to evaluate their adaptative responses to starvation. The starved cells showed an altered biochemical and enzymatic profiles, respectively, on Api 20E and Api ZYM systems and an evolution to the filterable minicells state capable to pass membrane pore size 0.45 μm. Outer membrane proteins patterns of stressed bacteria were also altered. Indeed, these modifications were manifested by the appearance and/or disappearance of bands as well as in the level of expression of certain proteins. Plasmids profiles analysis showed that V. alginolyticus ATCC 33787 lost three plasmids, whereas other tested strains conserved their initial profiles.     

Exogenous maltose enhances Zebrafish immunity to levofloxacin-resistant Vibrio alginolyticus

Understanding the interplay between bacterial fitness, antibiotic resistance, host immunity and host metabolism could guide treatment and improve immunity against antibiotic-resistant pathogens. The acquisition of levofloxacin (Lev) resistance affects the fitness of Vibrio alginolyticus in vitro and in vivo. Lev-resistant (Lev-R) V. alginolyticus exhibits slow growth, reduced pathogenicity and greater resistance to killing by the host, Danio rerio (zebrafish), than Lev-sensitive (Lev-S) V. alginolyticus, suggesting that Lev-R V. alginolyticus triggers a weaker innate immune response in D. rerio than Lev-S V. alginolyticus. Differences were detected in the metabolome of D. rerio infected with Lev-S or Lev-R V. alginolyticus. Maltose, a crucial metabolite, is significantly downregulated in D. rerio infected with Lev-R V. alginolyticus, and exogenous maltose enhances the immune response of D. rerio to Lev-R V. alginolyticus, leading to better clearance of the infection. Furthermore, we demonstrate that exogenous maltose stimulates the host production of lysozyme and its binding to Lev-R V. alginolyticus, which depends on bacterial membrane potential. We suggest that exogenous exposure to crucial metabolites could be an effective strategy for treating and/or managing infections with antibiotic-resistant bacteria.     

In-vitro anti-<Emphasis Type="Italic">Vibrio</Emphasis> spp. activity and chemical composition of some Tunisian aromatic plants

The chemical composition of five aromatic plants (Mentha longifolia, M. pulegium, Eugenia caryophyllata, Thymus vulgaris and Rosmarinus officinalis) frequently used in food preparation in Tunisia was analysed by GC-MS. The antimicrobial effect of the essential oils obtained from these plants was tested against Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio fluvialis strains. Thyme oil exhibited a high level of antimicrobial activities against Vibrio spp. strains. The diameter of the zones of growth inhibition for V. parahaemolyticus species was interestingly high (ranging from 14.66 to 28 mm). The MIC and MBC values were interestingly low for thyme oil (MIC 0.078–0.156 mg/ml) and (MBC >0.31–1.25 mg/ml). These results showed that these plants especially thyme and clove, can be to be used for seafood preparation to protect against contamination by Vibrio spp. strains. An erratum to this article can be found at     

Loop‐mediated isothermal amplification method for rapid detection of Vibrio alginolyticus,the causative agent of vibriosis in mariculture fish

Aims: The purpose of this study was to develop a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive and simple detection of Vibrio alginolyticus in mariculture fish. Methods and Results: LAMP primers were designed by targeting the gyrB gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64°C in a simple water bath. The detection sensitivity of the LAMP assay for the detection of V. alginolyticus is about 3·7 × 10² CFU ml^?1 (3·7 CFU per reaction). LAMP products could be judged with agar gel or naked eye after the addition of SYBR Green I. There were no cross‐reactions with other bacterial strains indicating a high specificity of the LAMP. The LAMP method was applied to detect V. alginolyticus‐infected fish tissues effectively. Conclusions: The LAMP established in this study is a simple, sensitive, specific, inexpensive and rapid protocol for the detection of V. alginolyticus. Significance and Impact of the Study: This LAMP method provides an important diagnostic tool for the detection of V. alginolyticus infection both in the laboratory and field.     

Distribution of some virulence related-properties of Vibrioalginolyticus strains isolated from Mediterranean seawater (Bay of Khenis, Tunisia): investigation of eight Vibriocholerae virulence genes

This study characterizes 28 Vibrio alginolyticus strains isolated from seawater from the Seacoast of Monastir (Khenis; Tunisia). V. alginolyticus were isolated using the TCBS modified agar plates and the biochemical activities were tested using RapID NF plus Strips. Proteases activities, hemolysis, antibiotics susceptibility, and adhesion to fish mucus and epithelial cell lines (Hep-2 and Caco-2) were also investigated. Eight Vibrio cholerae virulence genes (toxR, toxS, toxRS, toxT, ctxA, vpi, ace, zot) were investigated by PCR in genomes of V. alginolyticus strains. Most of the studied strains were β-haemolytic and produce many proteolytic enzymes. All isolates described here were resistant to several antibiotics tested. Six strains were able to adhere strongly to both Hep-2 and Caco-2 cell lines. The PCR investigation of V. cholerae genes showed a large distribution among the genomes of all V. alginolyticus strains. The toxR operon was found in 9 V. alginolyticus strains out of 28 studied. Only one strain was positive for the toxS and toxRS respectively. Five strains showed a positive amplification for the virulence pathogenic island (vpi), seven for the toxT, 3 for the ctxA and 9 for the Zonula occludens toxin (zot). The bay of Khenis harbors different genotypes of V. alginolyticus strains who inheritated several virulence genes from autochthones bacteria such as V. cholerae. These strains were able to produce several virulence enzymes and exhibit a high power to adhere to human epithelial cells and fish mucus.     

Expression and immunogenicity analysis of accessory colonization factor A from Vibrio alginolyticus strain HY9901

The accessory colonization factor A (ACFA) of Vibrio alginolyticus plays an important role in the efficient colonization of the bacterium and is potential candidates for vaccine development. In present study, the acfA gene was cloned, expressed and purified. Western blot analysis revealed protein recognition with the native ACFA in different V. alginolyticus strains. To analyze the immunogenicity of the recombinant ACFA, Lutjanus erythropterus Bloch were immunized by intraperitoneal injection, and the results demonstrated that the recombinant ACFA produced an observable antibody response in all sera of the vaccinated fish. The differential expressions of RAG1 gene in various tissues of L. erythropterus were analyzed by fluorescent quantitative real-time PCR, and the results showed the RAG1 mRNA expression was significantly up-regulated in thymus, head kidney and spleen tissue. Furthermore, the protective property of recombinant ACFA was evaluated through challenge with six heterogeneous virulent V. alginolyticus strains, and the immunohistochemical analysis in different tissues after challenge with V. alginolyticus. The results showed L. erythropterus vaccinated with recombinant ACFA were more tolerant of the infection by virulent V. alginolyticus strains. The data indicate that the recombinant ACFA could provide heterologous protection for the different virulent V. alginolyticus strains.     

Cloning and characterization of the tiger shrimp lysozyme

Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections. A lysozyme gene isolated from tiger shrimp, Penaeus monodon, was cloned, sequenced and characterized. The cDNA consists of a signal peptide of 18 amino acids and a mature peptide of 140 amino acids. The lysozyme is presumed to be a chicken-type lysozyme for it possesses two catalytic sites and eight cysteine residues which are highly conserved across species of chicken-type lysozymes. The lysozyme cDNAs of Penaeus semisulcatus, Litopenaeus vannamei, Macrobrachium nipponense and Macrobrachium rosenbergii were also cloned. High similarities existed among shrimp and prawn lysozymes but phylogenetic relationship of shrimps and prawns based on lysozyme molecules did not quite consistent with traditional taxonomic classification. High mRNA expression was detected in hepatopancreas, haemocytes and gill of tiger shrimp. Recombinant lysozyme exhibited potent lytic activities against fish pathogens providing evidence of the involvement of lysozyme in shrimp immunity.     

Expression,purification and antibody preparation of flagellin FlaA from Vibrio alginolyticus strain HY9901

Aims: The main aims of this study were to clone and express flagellin flaA gene from Vibrio alginolyticus strain HY9901, also to prepare mouse anti‐FlaA polyclonal antibody for future pathogen or vaccine study. Methods and Results: The full‐length flaA gene was amplified by PCR with designed primers. The open reading frame of flaA gene contains 1131 bp, and its putative protein consists of 376 amino acid residues. Alignment analysis indicated that the FlaA protein was highly conserved. SDS–PAGE indicated that the FlaA protein was successfully expressed in Escherichia coli BL21 (DE3). Then, the recombinant FlaA protein was purified by affinity chromatography, and the mouse anti‐FlaA serum was produced. The expression of flaA gene was verified by various immunological methods, including western blotting, enzyme‐linked immunosorbent assay (ELISA) and immunogold electron microscopy (IEM). Conclusions: Flagellin flaA gene was cloned and identified from V. alginolyticus HY9901, the recombinant FlaA protein was expressed and purified, and high‐titre FlaA protein‐specific antibody was produced. Western blot analysis revealed that the prepared antiserum not only specifically react to FlaA fusion protein, but also to natural FlaA protein of V. alginolyticus. The expressed FlaA protein was demonstrated, for the first time, as the component of flagella from V. alginolyticus by IEM. Significance and Impact of the Study: This study may offer important insights into the pathogenesis of V. alginolyticus, provide a base for further studies on the diagnosis and evaluation that whether the FlaA protein could be used as an effective vaccine candidate against infection by V. alginolyticus and other Vibrio species. Additionally, the purified FlaA protein and polyclonal antibody can be used for further functional and structural studies.     

乌梅提取物对溶藻弧菌的抑制效应与机理

[背景] 溶藻弧菌（Vibrio alginolyticus）能够感染鱼、虾、贝等海洋经济动物,给海水养殖业带来了严重的经济损失,对公众健康及食品安全也构成较大威胁。[目的] 通过研究乌梅（Fructus mume）对溶藻弧菌的抑菌效应及其机理,为乌梅在水产养殖中的进一步开发利用提供理论依据。[方法] 采用试管二倍稀释法测定乌梅提取物（Fructus mume Extract,FME）对溶藻弧菌的最小抑菌浓度（Minimal Inhibitory Concentration,MIC）和最小杀菌浓度（Minimum Bactericidal Concentration,MBC）,电导率仪检测溶藻弧菌的相对电导率,分光光度法分析溶藻弧菌的核酸泄露和呼吸链脱氢酶活性及生物膜抑制率,蛋白质电泳检测溶藻弧菌的蛋白质合成情况,扫描电子显微镜观察溶藻弧菌的亚显微结构。[结果] 乌梅提取物对溶藻弧菌的MIC和MBC分别为1.953 mg/mL和3.906 mg/mL;乌梅提取物显著增加了溶藻弧菌的相对电导率和核酸泄露,明显降低了溶藻弧菌蛋白质的合成能力,对于弧菌的亚显微形态产生了较大影响。同时乌梅提取物显著抑制了溶藻弧菌生物膜的形成和呼吸链脱氢酶的活性。[结论] 乌梅提取物通过增加溶藻弧菌细胞膜通透性及显著抑制生物膜的生成、呼吸链脱氢酶活性及蛋白质的合成,实现抑制及杀灭溶藻弧菌的目的。     

MnHSP90 cDNA characterization and its expression during the ovary development in oriental river prawn, <Emphasis Type="Italic">Macrobrachium nipponense</Emphasis>

Heat shock protein 90 (HSP90) is not only involved in environmental stress but also plays roles in the ovary development in some vertebrates. To understand its role in crustacean, we examined the HSP90 cDNA for the first time in the ovary and hepatopancreas of the oriental river prawn, Macrobrachium nipponense and designated this protein as MnHSP90 in this study. The MnHSP90 was cloned by the methods of degenerated oligonucleotide primers and rapid amplification of the cDNA ends (RACE). Bioinformatics analysis showed that the MnHSP90 cDNA was 2,684 bp in length, containing a 126 bp 5′ untranslated region (UTR), a 359 bp 3′ UTR, and an open reading frame (ORF) of 2,199 bp encoding a 732-amino acid polypeptide with predicted molecular mass of 84.3 KDa. Sequence alignment showed that the MnHSP90 shared 72–79% identity with other animals. Real-time quantitative PCR (qPCR) analysis demonstrated that the MnHSP90 mRNA was ubiquitously detected in all tested tissues, with the highest expression in the thoracic ganglia, the mediate in heart, muscle and intestine, and the lowest in haemocytes and gills. The MnHSP90 mRNA levels in the hepatopancreas and ovary of M. nipponense reached a maximum at the stage III (early vitellogenic stage) and stage IV (later vitellogenic stage) ovaries, respectively, and then decreased significantly in both tissues as the ovarian development proceeded. The level of MnHSP90 expression in the hepatopancreas was higher than that in the ovary when compared with in the same ovarian developmental stage. Our results indicate that MnHSP90 is involved in ovarian development in oriental river prawn and may play a regulatory role in ovary maturation.     

Occurrence of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> in the German Bight over a seasonal cycle

Bacteria of the genus Vibrio are an important component of marine ecosystems worldwide. The genus harbors several human pathogens, for instance the species Vibrio parahaemolyticus, a main cause for foodborne gastroenteritis in Asia and the USA. Pathogenic V. parahaemolyticus strains emerged also in Europe, but little is known about the abundance, pathogenicity and ecology of V. parahaemolyticus especially in Northern European waters. This study focuses on V. parahaemolyticus and its close relative Vibrio alginolyticus in the North Sea (Helgoland Roads, Germany). Free-living, plankton-attached and shellfish-associated Vibrio spp. were quantified between May 2008 and January 2010. CFUs up to 4.3 × 10³ N l⁻¹ and MPNs up to 240 N g⁻¹ were determined. Phylogenetic classification based on rpoB gene sequencing revealed V. alginolyticus as the dominant Vibrio species at Helgoland Roads, followed by V. parahaemolyticus. We investigated the intraspecific diversity of V. parahaemolyticus and V. alginolyticus using ERIC-PCR. The fingerprinting disclosed three distinct groups at Helgoland Roads, representing V. parahaemolyticus, V. alginolyticus and one group in between. The species V. parahaemolyticus occurred mainly in summer months. None of the strains carried the virulence-associated genes tdh or trh. We further analyzed the influence of nutrients, secchi depth, temperature, salinity, chlorophyll a and phytoplankton on the abundance of Vibrio spp. and the population structure of V. parahaemolyticus. Spearman Rank analysis revealed that particularly temperature correlated significantly with Vibrio spp. numbers. Based on multivariate statistical analyses we report that the V. parahaemolyticus population was structured by a complex combination of environmental parameters. To further investigate these influences is the key to understanding the dynamics of Vibrio spp. in temperate European waters, where this microbial group and especially the pathogenic species, are likely to gain in importance.     

A tandem-repeat galectin involved in innate immune response of the pearl oyster Pinctada fucata

The cDNA of a tandem-repeat galectin from the pearl oyster Pinctada fucata (designated PfGal) was cloned by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of PfGal was 1386 bp, consisting of a 5′ untranslated region (UTR) of 26 bp, a 3′ UTR of 313 bp, and an open reading frame (ORF) of 1047 bp encoding a polypeptide of 348 amino acids with a predicted molecular weight of 38.09 kDa and theoretical isoelectric point of 8.49. Similar to other tandem-repeat galectins, PfGal contained two tandem carbohydrate recognition domains (CRDs), with typical conserved motifs which were important for carbohydrate recognition, and it appeared to possess neither a signal peptide nor a transmembrane domain. Fluorescent quantitative real-time PCR analyses indicated that PfGal mRNA was highly expressed in hemocytes, digestive gland and mantle, and its expression was increased in all studied tissues after Vibrio alginolyticus challenge. The temporal expression of PfGal mRNA in hemocytes challenged by V. alginolyticus was clearly time-dependent and reached the maximum level at 6 h post-challenge, and then recovered to the original level. These results collectively indicated that PfGal may be involved in the immune response against bacterial infection and clearance of bacterial pathogens in P. fucata.     

Molecular analysis of the QM gene from <Emphasis Type="Italic">Penaeus monodon</Emphasis> and its expression on the different ovarian stages of development

In present study, a QM gene was obtained from the ovary and neurosecretory organ in eyestalk cDNA library of black tiger prawn (Penaeus monodon). The full-length black tiger prawn QM (PmQM) cDNA contained a 5′-UTR of 41 bp, an ORF of 663 bp encoding a polypeptide of 220 amino acids with molecular weight 25.5 kDa, and a 3′-UTR of 54 bp. Homology analysis of the deduced amino acid sequence of the PmQM with other known QM sequences by MatGAT software revealed that the PmQM was high homology with other invertebrates. A conserved signature sequence of the QM family was found in the PmQM deduced amino acid sequence. Analysis of the tissue expression pattern of the PmQM gene showed that the PmQM mRNA was expressed in all tissues tested, with highest levels in ovary. Furthermore, the PmQM expression was found to be different in three important ovarian stages of development. The results indicated PmQM might play an important role in ovarian development.     

Immune parameters in the humoral fluids of amphioxus Branchiostoma belcheri challenged with Vibrio alginolyticus

The knowledge concerning the humoral immunity is scarce in amphioxus Branchiostoma belcheri. This study measured the humoral parameters including lysozyme, antimicrobial activity, microbial agglutinin and haemagglutinin in amphioxus humoral fluids before and after Vibrio alginolyticus challenge. After challenged with V. alginolyticus, the lysozyme activity, growth inhibiting activities against Escherichia coli and V. alginolyticus and microbial agglutinating activities against Micrococcus lysodeikticus, Bacillus subtilis and Staphylococcus aureus were all increased significantly and haemagglutinating activities against rabbit and human A and O erythrocytes in the humoral fluids were all increased earlier. In contrast, the agglutinating activities against Vibrio harvey and E. coli in the humoral fluids were reduced in response to V. alginolyticus challenge and the haemagglutinating activity against human B erythrocytes increased later.