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.     

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.     

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.     

基于核酸适配体的PCR法检测溶藻弧菌及其灭活菌

溶藻弧菌(Vibrio alginolyticus)分布广,数量多,发病率高,是水产养殖中常见的条件致病菌,而对溶藻弧菌进行快速准确的识别鉴定是其病害防治的前提和基础。核酸适配体,因为具有较高的亲和特异性,在微生物的识别鉴定方面展现出了巨大的优势。本文利用核酸适配体和适配体筛选产物,通过结合、洗涤、加热分离、PCR扩增以及电泳检测等步骤,对溶藻弧菌进行了检测鉴定。结果表明,适配体和筛选产物都能对溶藻弧菌及其灭活菌进行较好的识别鉴定,适配体筛选产物对溶藻弧菌的检测下限为10~3cfu/mL,而对其灭活菌的检测下限为10~2cfu/mL,适配体对溶藻弧菌及其灭活菌的检测下限都可达到10 cfu/mL。该方法对溶藻弧菌有较好的亲和特异性,并能较好地区分溶藻弧菌与哈维氏弧菌等水产常见病原菌,在水产病害的检测中显示了较好的应用前景。     

High‐throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I‐based real‐time PCR combined with melting curve analysis

This work describes a primer pair and a high‐throughput SYBR Green I‐based real‐time PCR protocol combined with melting curve analysis for identification and quantification of Vagococcus salmoninarum in bacterial cultures and infected fish tissues. The 16S rRNA gene was selected for the design of the primer pair (SalF and SalR). The sensitivity and specificity of this primer pair were compared with other previously designed for conventional PCR. Although both primer pairs showed 100% specificity using pure bacterial cultures or DNA extracted from bacteria or fish tissues, the primer pairs designed in this study showed the highest sensitivity with a detection limit of 0.034 × 10⁰ amplicon copies per assay (equivalent to 2 × 10^?11 ng/µl, C_q value of 30.49 ± 1.71). The developed qPCR protocol allowed the detection of V. salmoninarum in non‐lethal and lethal fish samples with detection levels of 0.17 × 10⁰ gene copies in tissues artificially infected and 0.02 × 10⁰ in tissues of fish experimentally infected with V. salmoninarum. The high sensitivity of the developed method suggests that it could be considered as a useful tool for diagnosis of vagococcosis and the detection of V. salmoninarum in asymptomatic or carrier fish.     

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.     

Development of real‐time and quantitative QCM immunosensor for the rapid diagnosis of Aeromonas hydrophila infection

Since bacterial infection cause a significant economic loss in fish farms, it is necessary to develop rapid diagnostic tools. Interests on label‐free biosensors have been raised for the rapid detection of aquatic pathogenic bacteria but have not been extensively studied yet. Here we report a quartz crystal microbalance (QCM) immunosensor system for the rapid and simple detection of Aeromonas hydrophila, a pathogen for fish and human, in comparison with a conventional indirect ELISA method. In QCM immunosensor system, an antibody against A. hydrophila was covalently cross‐linked to the gold surface of sensor chip and bacterial attachment was monitored as real‐time frequency shifts within 5 min. The frequency shifts were very positively related to the amounts of bacterial cells between 6.25 and 100 μg corresponding to 6 × 10⁶ to 10⁸ CFU with a high specificity. The QCM immunosensor was also able to detect bacterial cells in fish tissue extract in a dose‐dependent manner. Indirect ELISA also showed the dose‐dependent reaction and the amplified signal may allow a lower detection limit. However, QCM immunosensor system showed a more linear and reliable standard curve with R² value of almost 1 (0.9999). Moreover, detection of the bacteria was much quicker and simpler.     

Development of a colloidal gold immunochromatographic assay (GICA) for the rapid detection of Spiroplasma eriocheiris in commercially exploited crustaceans from China

Spiroplasma eriocheiris is an emerging pathogen in freshwater crustaceans. In recent years, Eriocheir sinensis, Procambarus clarkii, Litopenaeus vannamei, Macrobrachium rosenbergii and Macrobrachium nipponense had been infected by this pathogen in China. An immunochromatographic strip test using gold nanoparticles was developed for rapidly detecting this pathogen. The strip test based on the principle of sandwich immunoassay by the specific combination between the pathogen and polyclonal antibody on a nitrocellulose membrane. Positive samples were displayed as red lines at the test and control zones of the nitrocellulose membrane, while negative samples resulted in a red line only at the control zone. The limit of detection was proved to be 10⁶ Color Change Unit/ml. The test strip could be visually detected within 15 min and do not have cross‐reaction with other aquatic bacteria. This test strip allows on‐site rapid detection of S. eriocheiris in crustacean without the requirement of specialized equipment and professional personnel. The one‐step test strips developed in our study had high sensitivity, specificity, reproducibility and stability. In conclusion, this method was proved to be convenient, feasible, rapid and effective for detecting S. eriocheiris.     

Histopathological changes in giant freshwater prawn Macrobrachium rosenbergii (de Man 1879) fed with probiotic Bacillus licheniformis upon challenge with Vibrio alginolyticus

The effect of dietary supplementation of probiotic bacterium Bacillus licheniformis on the histopathological changes in Macrobrachium rosenbergii juveniles (4.0 ± 0.02 g) challenged with known pathogenic strain of Vibrio alginolyticus are reported. Two isocaloric basal diets supplemented with probiotic bacteria B. licheniformis (1.0 × 10⁹ cfu/g feed) and other without probiotic supplementation were fed to the M. rosenbergii juveniles for 45 days. The histological observations revealed no significant changes in the hepatopancreas and gut tissues of both the experimental and the control groups which indicate that the present bacterium is a safe candidate probiont for the host. Prawns were challenged with V. alginolyticus after 45 days of feeding with probiotic diet. The histopathological studies of the hepatopancreas revealed that M. rosenbergii fed with probiotic‐supplemented diet showed less changes as compared to the prawns fed with control diet on second and fourth day of post‐experimental challenge with V. alginolyticus. Histopathological observations revealed that the gills of the prawns fed with control diet were severely affected in comparison to the prawns fed with probiotic‐supplemented diet after challenging with V. alginolyticus. Results from this study revealed the improved protection by dietary incorporation of B. licheniformis in reducing the histopathological manifestations due to V. alginolyticus infection in freshwater prawn.     

Development of a real‐time PCR assay for detection and quantification of Streptococcus iniae using the lactate permease gene

The aim of this study is the development and evaluation of a rapid and accurate quantitative PCR (qPCR)‐based protocol for detection of zoonotic pathogen Streptococcus iniae in bacterial cultures and tissues of diseased fish. For this purpose, the lactate permease‐encoding (lldY) gene was selected as a target for the design of S. iniae‐specific primers based on comparative genomic analysis using 45 sequences retrieved from NCBI genome database. Specificity and applicability of these primers were tested using 115 bacterial strains and fish tissues infected with S. iniae. Sensitivity, reproducibility and efficiency of qPCR assay were also determined. The developed qPCR assay showed 100% specificity with pure bacterial cultures or DNA extracted from S. iniae or tissues of fish infected with the bacterium. The method has high sensitivity with a detection limit of 1.12 × 10¹ amplicon copies per assay (equivalent to 2 × 10^–9 ng/µl) using bacterial DNA and of 1.44 × 10¹ gene copies in tissues of fish infected with S. iniae. In conclusion, this qPCR protocol provides an accurate and sensitive alternative for the identification of S. iniae and its detection on fish tissues that can be implemented as a routine tool in microbiological laboratories.     

The use of diatom Skeletonema costatum on aquaculture‐produced purple sea urchin (Paracentrotus lividus) larvae and post‐larvae diet

The quality of the microalgae provided on Paracentrotus lividus larvae rearing is a primordial factor having a direct (nutritional properties) and indirect (water quality) impact on growth, competence and survival. Skeletonema costatum is a diatom commonly used in the bivalve cultivation. However, the use of this diatom in P. lividus larval cultivations is poorly known. The Rhodomonas spp. is a microalgae commonly used in sea urchin larvae culture. Three different diets were tested on P. lividus larvae and post‐larvae cultivation (D1—Rhodomonas marina, D2—S. costatum, D3—mixture of both algae). Larvae fed with the D2 diet (55.8%) and D3 (39.9%) had a survival at 15 DAH higher than D1 (5.5%). The low survival in D1 could be due to the higher microbiological load on microalgae (Vibrio alginolyticus and V. pectenicide). Larvae fed with S. costatum (D2) showed a lower development than other diets. The competency index was lower for larvae fed with the D2. These results show that microalgae diversified diets contribute to a better development of P. lividus larvae. During the settlement and post‐settlement phase, there was also a lower growth of the sea urchin fed with the D2 and a higher survival for D3.     

Polyclonal antibody‐based farmer‐friendly flow‐through test for the detection of acute hepatopancreatic necrosis disease in shrimp

Early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) is currently the most significant disease of shrimp in farms of Vietnam, Thailand, Malaysia, China and Mexico, and there is a great risk that it may spread to other shrimp farming countries. Although, an array of sophisticated detection tools for AHPND available, there is a need for a sensitive, simple and rapid detection method. In this study, a simple, sensitive, rapid and polyclonal antibody‐based farmer‐friendly flow‐through assay (FTA) test has been developed for the detection of AHPND pathogen. The recombinant Photorhabdus insect‐related (Pir) A toxin‐like protein of AHPND pathogen was used to immunize rabbits at 21‐day interval observed for highest antibody titre after third booster by ELISA. The raised rabbit antiserum was purified by affinity chromatography and characterized by Western blot. The antiserum showed no cross‐reactivity with AHPND‐free Vibrio parahaemolyticus, V. anguillarum, White Spot Virus (WSV), Aeromonashydrophila and Aphanomycesinvadans. This polyclonal rabbit antiserum was used to develop a farmer‐friendly FTA test for the detection of AHPND pathogen. This simple FTA testis is more sensitive and could detect PirA^VP toxin up to 0.121 µg/ml, compared with 0.242 µg/ml by immunodot assay. Furthermore, FTA test requires only 8–10 min for completion, compared with 3 hr by immunodot thus found to be more sensitive, specific and cost‐effective. Collectively, sensitive FTA test would help shrimp farmers to take real‐time management decisions, especially emergency harvest and finally be a better hope for the prevention of AHPND.     

Enhanced immune responses and effectiveness of refined outer membrane protein vaccines against Vibrio harveyi in orange‐spotted grouper (Epinephelus coioides)

Vibriosis is a severe infection occurring in many commercially important marine fish species. In this study, vaccines containing Vibrio harveyi recombinant outer membrane protein K (rOmpK), outer membrane protein U (rOmpU) and rOmpK‐OmpU fusion protein in addition to the metabolizable Montanide^TM ISA 763 A VG adjuvant were developed and evaluated in the orange‐spotted grouper. The results indicate that recombinant V. harveyi protein‐based vaccines resulted in a remarkably higher expression of IL‐1β and IL‐8 at 24 hr, and greater antibody production, as early as 2 weeks postimmunization. Notably, enhanced immune responses and significant protective efficacy against V. harveyi infections were observed in the fusion protein vaccine‐injected fishes with relative per cent survival value of 81.8%. Additionally, the rOmpK‐OmpU antisera presented a high bactericidal effect on not only V. harveyi, but also Vibrio parahaermolyticus and Vibrio alginolyticus. Our results demonstrated that the fusion protein rOmpK‐OmpU was an effective vaccine candidate that exhibited potentially great versatility for controlling vibrio infections.     

Putative apolipoprotein A‐I,natural killer cell enhancement factor and lysozyme g are involved in the early immune response of brown‐marbled grouper,Epinephelus fuscoguttatus,Forskal, to Vibrio alginolyticus

The gram‐negative bacterium, Vibrio alginolyticus, has frequently been identified as the pathogen responsible for the infectious disease called vibriosis. This disease is one of the major challenges facing brown‐marbled grouper aquaculture, causing fish farmers globally to suffer substantial economic losses. The objective of this study was to investigate the proteins involved in the immune response of brown‐marbled grouper fingerlings during their initial encounter with pathogenic organisms. To achieve this objective, a challenge experiment was performed, in which healthy brown‐marbled grouper fingerlings were divided into two groups. Fish in the treated group were subjected to intraperitoneal injection with an infectious dose of V. alginolyticus suspended in phosphate‐buffered saline (PBS), and those in the control group were injected with an equal volume of PBS. Blood samples were collected from a replicate number of fish from both groups at 4 h post‐challenge and analysed for immune response‐related serum proteins via two‐dimensional gel electrophoresis. The results showed that 14 protein spots were altered between the treated and control groups; these protein spots were further analysed to determine the identity of each protein via MALDI‐TOF/TOF. Among the altered proteins, three were clearly overexpressed in the treated group compared with the control; these were identified as putative apolipoprotein A‐I, natural killer cell enhancement factor and lysozyme g. Based on these results, these three highly expressed proteins participate in immune response‐related reactions during the initial exposure (4 h) of brown‐marbled grouper fingerling to V. alginolyticus infection.     

Indigenous AHL‐degrading bacterium Bacillus firmus sw40 affects virulence of pathogenic Aeromonas hydrophila and disease resistance of gibel carp

Interrupting quorum sensing represents a novel anti‐infective strategy to combat bacterial pathogen, and biodegradation of quorum sensing signal AHLs has been proved to be an efficient way to control pathogenic Gram‐negative bacteria in aquaculture. In this study, the effect of Bacillus firmus sw40 as efficient AHL‐degrading strain on virulence of fish pathogen Aeromonas hydrophila and disease resistance of gibel carp Carassius auratus gibelio was investigated. The results demonstrated that in vitro the B. firmus sw40 extracellular production (ECP) was able to significantly decrease protease production, haemolytic activity and biofilm formation in A. hydrophila. Dietary administration of B. firmus sw40 (10⁹ CFU/g) for 4 weeks significantly reduced the inflammatory cytokines TNF‐1a, TNF‐2a and IFN‐γ genes expression, antioxidant parameter MDA and GSH levels in serum and increased antioxidant enzyme SOD activity. Besides, B. firmus sw40 could significantly increase the survival of gibel carp with pathogenic A. hydrophila infection.     

Bench‐top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture

No gold standard assay exhibiting error‐free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non‐culture assays in three matrices (phosphate‐buffered saline, ovarian fluid and kidney tissue). Non‐culture assays included polyclonal enzyme‐linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane‐filtration FAT, nested polymerase chain reaction (nested PCR) and three real‐time quantitative PCR assays. Injection challenge of specific pathogen‐free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.     

Vibrogen‐2 vaccine trial in lumpfish (Cyclopterus lumpus) against Vibrio anguillarum

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen‐2 is a commercial polyvalent bath vaccine that contains formalin‐inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen‐2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath‐immunized, bath‐boost‐immunized at four weeks post‐primary immunization, and intraperitoneally (i.p.) boost‐immunized at eight weeks post‐primary immunization. The control groups were i.p. mock‐immunized with PBS. Twenty‐seven weeks post‐primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD₅₀ dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post‐challenge. Commercial vaccine Vibrogen‐2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.     

Rapid and sensitive detection of Vibrio harveyi by loop‐mediated isothermal amplification combined with lateral flow dipstick targeted to vhhP2 gene

Vibrio harveyi is a causative agent of the Vibriosis or luminescent bacterial disease in worldwide aquaculture industry. A reliable assay for identification of V. harveyi infection is important to prevent the bacterial spread. In this study, biotinylated loop‐mediated isothermal amplification (LAMP) amplicons were produced by a set of four designed primers that recognized specifically the V. harveyi vhhP2 gene, encoding a putative outer membrane protein with unknown function, followed by hybridization with an fluorescein isothiocyanate (FITC)‐labelled probe and lateral flow dipstick (LFD) detection. A novel set of PCR primer was also designed specifically to vhhP2 gene and appear to be a species‐specific tool for V. harveyi detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP‐LFD and PCR methods accurately identified 22 isolates of V. harveyi but did not detect 16 non‐harveyi Vibrio isolates, and 34 non‐Vibrio bacterial isolates. The sensitivity of LAMP‐LFD for V. harveyi detection in pure culture was 1.1 × 10² CFU mL^?1 or equivalent to 0.6 CFU per reaction, while that of PCR was 6 CFU per reaction. For spiked shrimp sample, the sensitivity of LAMP was 1.8 × 10³ CFU g^?1 or equivalent to 5 CFU per reaction, while that of PCR was 50 CFU per reaction. In conclusion, the established LAMP‐LFD methods provided a valuable tool for rapid identification of V. harveyi and can be used to distinguish V. harveyi from V. campbellii.     

Identification of novel immunogenic proteins of Vibrio alginolyticus by immunoproteomic methodologies

Vibrio alginolyticus is one of the most serious diseases in cultured marine and freshwater fish and shellfish. The absence of suitable vaccine or virulent marker can be the bottleneck to control V. alginolyticus infection. In this study, immunoproteomic approaches were undertaken to study the immunogenicity of the whole‐cell protein of V. alginolyticus HY9901. The whole‐cell proteins were analysed by two‐dimensional gel electrophoresis and subsequent immunoblotting using the rabbit anti‐V. alginolyticus HY9901 serum. A total of 55 immunogenic proteins were identified by immunoproteomic analysis. Of the 55 proteins, 51 are specific immunoreactive proteins and four are nonspecific immunoreactive proteins. Furthermore, outer membrane protein N (spot 2) was used as immunogens to immunize Epinephelus coioides for investigation of protective abilities and activities. The E. coioides immunized with OmpN has abilities to fight against infections caused by V. alginolyticus. The other novel immunogenic proteins may be developed as alternative antigens for further study of V. alginolyticus vaccine and diagnostics. These data show that immunoproteomics methods can be successfully applied in identifying immunogenic proteins of V. alginolyticus, which helps to search for the protective antigens in future.