The role of rpoS in the regulation of Vibrio alginolyticus virulence and the response to diverse stresses

Vibrio alginolyticus is a leading aquatic pathogen, causing huge losses to aquaculture. rpoS has been proven to play a variety of important roles in stress response and virulence in several bacteria. In our previous study, upon treatment with Cu²⁺, Pb²⁺, Hg²⁺ and low pH, the expression levels of rpoS were downregulated as assessed by RNA‐seq, while impaired adhesion ability was observed, indicating that rpoS might play roles in the regulation of adhesion. In the present study, the RNAi technology was used to knockdown rpoS in V. alginolyticus. In comparison with wild‐type V. alginolyticus, RNAi‐treated bacteria showed significantly impaired abilities of adhesion, growth, haemolytic, biofilm production, movement and virulence. Meanwhile, alterations of temperature, salinity, pH and starvation starkly affected rpoS expression. The present data suggested that rpoS is a critical regulator of virulence in V. alginolyticus; in addition, rpoS regulates bacterial adhesion in response to temperature, pH and nutrient content changes. These are helpful to explore its pathogenic mechanism and provide reference for disease control.     

Cloning,expression of Vibrio alginolyticus outer membrane protein‐OmpU gene and its potential application as vaccine in crimson snapper,Lutjanus erythropterus Bloch

The outer membrane proteins of the marine aquatic animal pathogen, Vibrio alginolyticus, play an important role in the virulence of the bacterium and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein‐OmpU was cloned and expressed in Escherichia coli. Polyclonal antibodies were raised in rabbits against the purified recombinant OmpU, and the reaction of the antibody was confirmed by Western blotting using the isolated OmpU and the recombinant OmpU of V. alginolyticus. To analyze the immunogenicity of the recombinant OmpU, crimson snapper, Lutjanus erythropterus Bloch, were immunized by intraperitoneal injection, and antibody response was assessed by the enzyme‐linked immunosorbent assay (ELISA). The results demonstrated that the recombinant OmpU produced an observable antibody response in all sera of the vaccinated fish. The vaccinated fish were challenged by virulent V. alginolyticus and observed to have high resistance to infection. These results indicate that the recombinant OmpU is an effective vaccine candidate against V. alginolyticus in L. erythropterus.     

Selection and characterization of ssDNA aptamers specifically recognizing pathogenic Vibrio alginolyticus

Vibrio alginolyticus (V. alginolyticus) is a major opportunistic pathogen to both marine animals and humans, which has also caused heavy economic losses to mariculture. The aim of this study was to develop highly specific aptamers for V. alginolyticus. Single‐stranded DNA (ssDNA) aptamers with high binding affinity to viable V. alginolyticus were generated by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and identified by flow cytometric analysis in this study. The selected aptamers showed high specificity for V. alginolyticus and low apparent binding for other bacteria. The aptamers formed distinct stem‐loop structures, which could form the basis of aptamers’ specific binding to the target V. alginolyticus. Aptamer VA2 and VA8 showed particularly high binding affinity constant (Kd) of 14.31 ± 4.26 and 90.00 ± 13.51 nM, respectively. The aptamers produced no cytotoxic effects in vitro and in vivo. ssDNA aptamers were successfully selected against the viable bacteria pathogen V. alginolyticus by SELEX. The aptamers selected in this study could be not only applied as specific chemical molecular probes for studying V. alginolyticus pathogenesis to Trachinotus ovatus, but also developing rapid convenient diagnosis assay for V. alginolyticus infection, even when applied to the complex sample matrix, such as food and environment samples.     

Effects of crude extracellular protein and crude intracellular polysaccharides of Vibrio alginolyticus on the growth,energy metabolism regulation and WSSV resistance of Litopenaeus vannamei

This study aimed to investigate the effects of adding crude extracts of the extracellular protein (Ex‐Pro) and intracellular polysaccharides (In‐Poly) of Vibrio alginolyticus to diets, at a dosage of 10 g/kg, on the growth, energy metabolism and WSSV (White Spot Syndrome Virus) resistance of Litopenaeus vannamei (initial weight, 0.88 ± 0.04 g/shrimp). Growth and survival rate were not significantly affected by the crude extracts (p > 0.05). Shrimp fed Ex‐Pro crude extracts showed higher succinate dehydrogenase (SDH) activity in the hepatopancreas and muscle but lower lactate dehydrogenase (LDH) activity in the muscle (p < 0.05). The activities of hexokinase (HK), pyruvate kinase (PK), LDH and SDH in the hepatopancreas and the activity of SDH in the muscle were significantly increased by feeding In‐Poly crude extracts (p < 0.05). In contrast, the content of fatty acid synthase (FAS) in the muscle was significantly reduced by the crude extracts (p < 0.05). The contents of glucose and triglyceride and the activity of the electron transport system in the hepatopancreas were significantly increased by the crude extracts (p < 0.05), and the WSSV resistance of the shrimp was increased. These results indicated that the Ex‐Pro and In‐Poly crude extracts of V. alginolyticus could affect energy metabolism, and there was a correlation between WSSV resistance and energy metabolism in L. vannamei.     

Development of novel aptamer‐based enzyme‐linked apta‐sorbent assay (ELASA) for rapid detection of mariculture pathogen Vibrio alginolyticus

As the major opportunistic pathogen to both marine animals and humans, Vibrio alginolyticus (V. alginolyticus) has caused heavy economic losses to mariculture. ssDNA aptamer VA2 targeting live V. alginolyticus was generated by systematic evolution of ligands by exponential enrichment (SELEX) technology in our previous study. In this study, we first developed aptamer (VA2)‐based enzyme‐linked apta‐sorbent assay (VA2‐ELASA) for rapid detection of mariculture pathogen V. alginolyticus. The VA2‐ELASA could achieve the rapid detection for V. alginolyticus infection with high specificity and sensitivity. The VA2‐ELASA could specifically identify V. alginolyticus, but not other non‐target bacterial strains. VA2‐ELASA could detect V. alginolyticus at the concentration of 5 × 10⁴/ml, the incubation time short to 1 min and the incubation temperature as high as 45°C, which proved sensitivity and stability of the novel VA2‐ELASA in this study. It took less than one hour to accomplish the detection process by VA2‐ELASA. The characteristics of specificity, sensitivity and easy operation make VA2‐ELASA a novel useful technology for the rapid diagnosis of pathogen V. alginolyticus in mariculture.     

Virulence‐associated factors in Vibrio cholerae non‐O1/non‐O139 and V. mimicus strains isolated in ornamental fish species

During recent decades, ornamental fish have proven to be one of the fastest growing categories of pets in Europe. In this framework, we evaluated both the potential pathogenic and zoonotic risks caused by 53 Vibrio cholerae non‐O1/non‐O139 and a Vibrio mimicus strain isolated from ornamental fish species mostly originating from South‐East Asia countries between 2000 and 2015 in Italy. All the strains were firstly identified at species level by biochemical, phylogenetic and mass spectrometry (matrix‐assisted laser desorption ionization time of flight) methods, and then studied to reveal the presence of the main virulence and colonization‐associated factors, as ctxA, ace, zot, stn/sto, toxR, rtxA, hlyA and tcpA by multiplex and single endpoint PCR assays. Findings showed that 21 of 54 strains harboured at least one virulence factor with a predominance for the toxR⁺, rtxA⁺ and hlyAET⁺ genotype. Interestingly, the V. mimicus strain harboured the colonization factor and the CTX prophage receptor, tcpA, indicating the ability to capture and integrate it in its genome increasing its pathogenicity. Although these enterotoxins can sporadically cause gastroenteritis, the results highlight their probable involvement in causing severe implications for public health, suggesting the need for an European microbiological monitoring.     

鳗弧菌、溶藻胶弧菌外膜蛋白的分离及特性

分别用SDS、Sarkosyl及PMSF法分离制备鳗弧菌（Vibrio anguillarum）、溶藻胶弧菌（Vibrio alginolyticus）主要外膜蛋白（MOMP），通过SDS-PAGE分析比较2种弧菌主要外膜蛋白的组成结构。结果表明，SDS、Sarkosyl和PMSF法分离提取的鳗弧菌和溶藻胶弧菌外膜蛋白中，SDS-PAGE电泳图谱不同，鳗弧菌外膜蛋白分子量为27-138kD；溶藻胶弧菌外膜蛋白分子量为27-104kD，其中，45kD、30kD和27kD外膜蛋白为鳗弧菌和溶藻胶弧菌所共有。经比较，Sarkosyl法对2种弧菌外膜蛋白的提取效果较好。对3种方法提取的2种弧菌的主要外膜蛋白进行SDS PAGE后，分别与吸附后的兔抗鳗弧菌、抗溶藻胶弧菌血清的Western-blotting印迹显示，兔抗鳗弧菌血清与其菌株的外膜蛋白主要有3条免疫反应带，其分子量分别为97kD、51kD和30kD，说明其可能与鳗弧菌抗原的特异性有关；兔抗溶藻胶弧菌血清与其菌株的外膜蛋白主要有2条免疫反应带，其分子量分别为51kD和27kD，说明可能与溶藻胶弧菌抗原的特异性有关，而51kD外膜蛋白可能是2种弧菌共有的特异性抗原。     

大菱鲆致病性溶藻弧菌SR1的外膜蛋白及其抗原性分析

用十二烷基肌氨酸钠(Sarkosyl)抽提结合超速离心的方法提取了一株大菱鲆致病性溶藻弧菌(Vibrio alginolyticus)SR1和其他7株弧菌的外膜蛋白。通过SDS-PAGE图谱分析比较了这8株弧菌外膜蛋白的组成,结果表明,8株弧菌的外膜蛋白电泳一般可得到6-12条条带,其分子量多集中在65-106 kD和28-48 kD,其中36 kD的蛋白带为8株弧菌所共有。用兔抗SR1全菌血清进行Western-blot印迹显示,菌株SR1的外膜蛋白条带中有6条发生了阳性反应,其分子量分别为73 kD、48 kD4、5 kD3、9 kD、36 kD和32 kD。而其他7株弧菌的外膜蛋白与兔抗SR1血清也发生程度不等的阳性反应,这些阳性反应条带的分子量集中在65-73 kD、45-48 kD和36-41 kD之间,其中36 kD的外膜蛋白在8株弧菌中均出现明显的阳性反应,说明36 kD的外膜蛋白是这8株弧菌共有的特异性抗原。     

Identification of novel sRNAs involved in oxidative stress response in the fish pathogen Vibrio alginolyticus by transcriptome analysis

Vibrio alginolyticus as an important pathogen in aquaculture can encounter the oxidative stress produced by the immune system during infection. Previous studies showed that sRNAs have important functions in response to oxidative stress in bacteria; however, less of sRNAs related to oxidative stress response were identified in V. alginolyticus. In this study, a total of 749 novel sRNAs were identified by RNA sequencing; among them, 128 sRNAs were up‐ or downregulated in response to oxidative stress. In addition, 1,870 genes exhibited variation on mRNA levels in oxidative stress response. By analysing the target genes of the sRNAs, we concluded that these sRNAs could regulate expressions of genes responsible for iron transport, catalase, GSH‐dependent defence system, electron transferred and stress response. Moreover, the functions of the sRNAs are also seemed related to the pathogenicity in V. alginolyticus. Based on the results, we constructed the oxidative stress model in V. alginolyticus. This study provides us the first outlook of sRNAs function in oxidative stress response in V. alginolyticus. Furthermore, this study can help us to prevent and control this important opportunistic pathogen in aquaculture.     

Detection of antigenic proteins expressed by lymphocystis virus as vaccine candidates in olive flounder, Paralichthys olivaceus (Temminck & Schlegel)

Although the major capsid proteins (MCPs) of lymphocystis disease virus (LCDV) have been characterized, little is known about the host-derived immune response to MCPs and other LCDV antigenic proteins. To identify antigenic proteins of LCDV that could be used as vaccine candidates in olive flounder, Paralichthys olivaceus, we analysed the viral proteins responsible for its virulence by applying immuno-proteomics. LCDV proteins were separated by one-dimensional gel electrophoresis, transferred to polyvinylidene difluoride membrane, and probed with homogeneous P. olivaceus antisera elicited by LCDV natural infection and vaccination with formalin-killed LCDV. Four immune-reactive proteins were obtained at 68-, 51-, 41- and 21 kDa using antisera collected from natural infection while two proteins at 51- and 21 kDa exhibited response to antisera from vaccinated fish, indicating that the latter two proteins have vaccine potential. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoelectrospray MS/MS, the 51 and 21 kDa proteins were identified as MCP and an unknown protein, respectively.     

Effects of Vibrio harveyi on plasma clotting protein (coagulogen) of white shrimp Litopenaeus vannamei

Blood clotting exhibits various important functions, including the prevention of body fluid loss and invasion of pathogens in shrimp. The effects of pathogenic Vibrio harveyi on plasma of white shrimp (Litopenaeus vannamei) in vitro and in vivo were investigated in this study. The clotting protein (coagulogen) in plasma of white shrimp pre‐incubated with extracellular products (ECP) of V. harveyi was found apparently decreased and fast‐migrated in crossed immunoelectrophoresis (CIE) gels. In addition, the coagulogen had been degraded to many low molecular‐weight protein bands in plasma pre‐incubated with ECP on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gels. When pre‐challenged with bacterial cells and ECP of V. harveyi, the white shrimp began to die at about 30 and 16 h respectively. Moreover, plasma coagulogen was decreased more obvious in shrimp challenged with ECP than that with bacterial cells as visualized in CIE gels, and total plasma protein in both group of shrimp were all decreased. Haemolymph withdrawn from moribund shrimp pre‐challenged with V. harveyi or its ECP was observed unclottable. However, the addition of clotting factors (transglutaminase and/or Ca²⁺) to these unclottable plasma could apparently promote their re‐clotting ability as jelly‐like solid observed in microtubes. The recovery of clotting ability of plasma from moribund shrimp was due to the reformation of coagulogen (200 kDa) after adding the two clotting factors as shown on CIE and SDS‐PAGE gels. The present results suggest that the infection of V. harveyi in white shrimp may not only degrade coagulogen but also influence the presence of transglutaminase and Ca²⁺ ion.     

Identification and typing of Vagococcus salmoninarum using genomic and proteomic techniques

This study reports on the characterization of Vagococcus salmoninarum using phenotypic, serological, antigenic, genetic and proteomic methods. All strains of V. salmoninarum were resistant to most of the antimicrobials tested, and only 10% of strains were sensitive to florfenicol. Serological analysis demonstrated a high antigenic homogeneity within the species. No cross‐reaction was detected with other fish pathogenic species causing streptococcosis (Lactococcus garvieae, Streptococcus parauberis, Streptococcus iniae, Streptococcus agalactiae, Carnobacterium maltaromaticum) using serum against V. salmoninarum CECT 5810. Electrophoretic analysis of cell surface proteins and immunoblot supported the antigenic homogeneity within V. salmoninarum strains. Moreover, limited diversity was detected using genomic (RAPD, ERIC‐PCR and REP‐PCR) and MALDI‐TOF‐MS analyses. The phenotypic, genomic and proteomic methods tested allowed the rapid differentiation of V. salmoninarum from the other species causing streptococcosis. However, MALDI‐TOF‐MS is the most promising method for typing and characterization of V. salmoninarum.     

Comparative assessment of Vibrio virulence in marine fish larvae

Vibrionaceae infections are a major obstacle for marine larviculture; however, little is known about virulence differences of Vibrio strains. The virulence of Vibrio strains, mostly isolated from vibriosis outbreaks in farmed fish, was tested in larval challenge trials with cod (Gadus morhua), turbot (Scophthalmus maximus) and halibut (Hippoglossus hippoglossus) using a multiwell dish assays with single‐egg/larvae cultures. The strains differed significantly in virulence as some caused a high mortality of larva reaching 100% mortality after a few days, while others had no or only marginal effects on survival. Some Vibrio strains were pathogenic in all of the larva species, while some caused disease only in one of the species. Twenty‐nine of the Vibrio anguillarum strains increased the mortality of larvae from at least one fish species; however, pathogenicity of the strains differed markedly. Other Vibrio species had no or less pronounced effects on larval mortalities. Iron uptake has been related to V. anguillarum virulence; however, the presence or absence of the plasmid pJM1 encoding anguibactin did not correlate with virulence. The genomes of V. anguillarum were compared (D. Castillo, P.W. D'Alvise, M. Middelboe & L. Gram, unpublished data) and most of the high‐virulent strains had acquired virulence genes from other pathogenic Vibrio.     

Limited performance of MALDI‐TOF for identification of fish Aeromonas isolates at species level

The aim of this study was to evaluate the usefulness of the MALDI‐TOF MS to identify 151 isolates of Aeromonas obtained mostly from diseased fish. MALDI‐TOF MS correctly identified all isolates to the genus level but important differences in the percentage of isolates correctly identified depending on the species were observed. Considering exclusively the first identification option, Aeromonas bestiarum, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas veronii and Aeromonas sobria were the best identified with results >95%. However, considering the first and second identification options, the only species that showed values >90% was A. hydrophila. Overall, when the database was supplemented with 14 new spectra, the number of accurate identifications increased (41% vs. 55%) and the number of inconclusive identifications decreased (45% vs. 29%), but great differences in the success of species‐level identifications were found. Species‐distinctive mass peaks were identified only for A. hydrophila and A. bestiarum (5003 and 7360 m/z in 95.5% and 94.1% of their isolates, respectively). This work demonstrates the utility of MALDI‐TOF MS for Aeromonas identification to the genus level, but there is no consistency for the accurate identification of some of the most prevalent species implicated in fish disease.     

Identification and characterization of pathogens associated with fin erubescence and ulceration of cultured Scophthalmus maximus

Scophthalmas maximus is a precious and main aquacultural species in northern China with an annual output value of more than 1.5 billion yuan. However, with the expansion of breeding range, increased stocking density and variety degradation, a variety of bacterial, parasitic and viral diseases have often occurred and worsened. Among them, bacterial disease is the most serious in farmed S. maximus. Since 2010, an epidemic bacterial disease with primary symptoms of lighter colour of lateral and caudal fins, spotty bleeding, fin base bleeding, abdominal swelling and redness and lesions of internal organs has put broken in S. maximus cultured in Weihai, Longkou and Haiyang, Shandong Province, China. The disease occurred mostly at the seedling and early development stages and propagated very fast, resulting in high mortality. These S. maximus died with obvious surface ulceration in only 3–4 days, causing great economic losses to the aquaculture industry. Beginning in 2010, we conducted epidemiological investigation in farmed S. maximus and isolated two different pathogens from the body of the diseased S. maximus from different farms. In this paper, we observed their morphology, conducted phylogenetic analysis based on their physiological and conventional biochemical characteristics and 16S rRNA gene sequences, preliminarily analysed their pathogenesis and investigated the histopathological changes of the diseased S. maximus. We believed that Vibrio anguillarum and V. parahaemolyticus were the pathogenic bacteria for the epidemic disease in S. maximus.     

Inhibition of Vibrio spp. by 2‐Hydroxyethyl‐11‐hydroxyhexadec‐9‐enoate of Marine Cyanobacterium Leptolyngbya sp. LT19

Seven marine cyanobacteria were isolated from two regions of the Gulf of Thailand and evaluated by the agar diffusion method for antibacterial activity. Inhibitory compound was purified from the crude methanol extract and its structure was elucidated based on extensive spectroscopic analysis, including IR, 1D and 2D NMR spectra as well as mass spectrometry. A novel antimicrobial compound produced by the marine cyanobacterium Leptolyngbya sp. LT19 was identified to be a 2‐hydroxyethyl‐11‐hydroxyhexadec‐9‐enoate which has so far never been reported in microorganisms. Biological assays revealed that this novel compound exhibited antibacterial activities against the Gram‐negative, persistent shrimp pathogens, Vibrio harveyi and V. parahaemolyticus with minimal inhibitory concentration of 250–1000 and 350–1000 μg mL^?1 respectively.