Salinity a significant environmental factor for Vibrio harveyi virulence in Fenneropenaeus indicus

The effect of ambient salinity on the haemolymph variables of Fenneropenaeus indicus and its susceptibility to Vibrio harveyi infection under salinity stress has been studied. Adult shrimps were acclimated to 5‰ (hypo osmotic), 25‰ (iso osmotic) and 35‰ (hyper osmotic) salinity levels and the animals were injected with a mid logarithmic culture of V. harveyi at sub lethal level and haemolymph parameters were analysed. Haemolymph proteins, intracellular superoxide anion production, phenoloxidase (PO), alkaline phosphatase (ALP) and acid phosphatase (ACP) activity were found to be at elevated level both at 5‰ and 35‰ post challenge. The haematological responses showed a progressive increase (P < 0.05) up to post challenge day 5 (PCD 5) followed by a considerable decline at all salinities with the lowest being at 35‰. The alterations in the variables were higher in shrimps held at 5‰. However, the V. harveyi infection was severe in animals held at 35‰. The reduction in the parameters could be correlated with the decrease in survival rate of shrimps at 35‰ with a concurrent increase in V. harveyi at this salinity. Multiple regression analysis revealed that ACP (P < 0.001), haemocyte protein HCP (P < 0.001) and PO (P < 0.05) could explain 91% variability in the shrimp survival. These parameters may be used as effective shrimp health indicators. It is evident from the study that ambient salinity alters the haemolymph variables, modulates the virulence in V. harveyi and makes the shrimps more vulnerable to infection at higher salinity. The virulence of V. harveyi is increased at 35‰ salinity as being evidenced from the high mortality at this salinity. The study emphasizes the importance of salinity as an important environmental factor both in terms of host susceptibility and virulence of the pathogen.     

Vibrio harveyi (VirB11) recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides)

Despite significant improvements in aquaculture to compensate wild catch, disease organisms have thrived in limiting its national and global potential. Using antibiotics, in a bid to remedy the havoc, has given rise to complications, attracting attention to disease prevention by immune enhancement against diseases. Grouper production has been inhibited for the threats of bacterial infection, particularly of Vibrio origin. Considering the rise in vibriosis cases, improved vaccines are necessary; moreover, recombinant vaccines, the choice for trial in the present experiment have been effective and more specific in improving immunity. The current work deals with grouper immune system enhancement with a recombinant vaccine developed from VirB11 gene in Vibrio harveyi. VirB11 was cloned in V. harveyi for recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides). As indicated by the results, recombinant VirB11 protein showed effectiveness in conferring protection against vibriosis with observable specific antibody response in enzyme‐linked immunosorbent assay (ELISA) analysis; a significant increase (p < 0.05) in antibody levels was observed after a week and after 8 weeks post‐vaccination. From the weeks post‐vaccination, log2 (antibody titres) in the sera of vaccinated groups reached a peak of 14.2 at week 5 in the vaccinated group in comparison with a peak of approximately 5 and 2 in adjuvant and PBS controls. As indicated by the challenge results, 90% relative survival was observed in vaccinated group and 13% relative survival in control group I (adjuvant control). The cumulative performance of protein concludes VirB11 commendable for recombinant vaccine development.     

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.     

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.     

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.     

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.     

Development of a two‐step,non‐probed multiplex real‐time PCR for surveilling Vibrio anguillarum in seawater

Vibrio anguillarum is an aggressive and halophilic bacterial pathogen most commonly originating from seawater. Vibrio anguillarum presence in fisheries and aquaculture facilities causes significant morbidity and mortality among aquaculture species primarily from haemorrhaging of the body and skin of the infected fish that eventually leads to death, collectively recognized as the disease vibriosis. This study served to develop a non‐probe, multiplex real‐time PCR assay to rapidly detect V. anguillarum presence in seawater. Specific primers targeting genes vah1, empA and rpoN of V. anguillarum were selected for multiplex reaction among 11 different primer sets and the extension step was eliminated. Primer concentration, denaturation time as well as annealing time and temperature of DNA amplification were optimized, thus reducing reaction duration. The two‐step, non‐probed multiplex real‐time PCR set forth by this study detects as little as 3 CFU mL^?1 of V. anguillarum presence in sea water, without enrichment cultivation, in 70 min with molecular precision and includes melting curve confirmation.     

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.     

Isolation,identification and pathogenicity of Vibrio harveyi,the causal agent of skin ulcer disease in juvenile hybrid groupers Epinephelus fuscoguttatus × Epinephelus lanceolatus

The hybrid grouper, Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂), is a newly bred cultivated marine fish species of high economic value. However, a skin ulcer disease with high mortality has occurred, and the responsible pathogen remains unknown. In this study, we summarized the epidemic status and external signs of this disease. We screened potential pathogens and finally isolated one bacterial strain ML01 from affected fish. We subjected healthy juvenile hybrid groupers to bacterial challenge tests with the isolate by immersion, immersion after dermal abrasion and intraperitoneal injection, respectively. Within 14 days post‐infection, the isolate ML01 caused mass mortality of juveniles infected via immersion after dermal abrasion or intraperitoneal injection. Diseased juveniles displayed obvious signs of skin ulcers. The median lethal dose of ML01 by intraperitoneal injection was 1.10 × 10⁵ colony‐forming units. ML01 was identified as Vibrio harveyi by bacterial morphology, analytical profile index identification, 16S rDNA sequencing and multilocus sequence analysis. Antibiotic susceptibility tests showed that ML01 was sensitive to ceftriaxone, doxycycline and minocycline. The results of this study suggest that V. harveyi is the causal agent of skin ulcer disease in juvenile hybrid groupers, thus providing a basis for effective control and prevention of this disease.     

Improvement of growth performance,water quality and disease resistance against Vibrio harveyi of postlarval whiteleg shrimp (Litopenaeus vannamei) by administration of mixed microencapsulated Bacillus probiotics

The objective of this study was to investigate the effects of mixed Bacillus on growth, water quality and disease resistance against Vibrio harveyi in whiteleg shrimp (Litopenaeus vannamei). Postlarval shrimp (PL₃₀) were fed with (a) a basal diet (the control), (b) a diet containing mixed freeze‐dried Bacillus probiotics (FB) and (c) addition of mixed microencapsulated Bacillus probiotics (MB) in culture water. Addition of FB and MB probiotics improved (p < .05) growth, feed efficiency, survival and culture water quality (ammonia and nitrite) compared to the control group although there was no difference (p > .05) between the two treated groups. Bacillus numbers in gastrointestinal tracts and culture water of FB‐ and MB‐administrated shrimp were higher (p < .05) than in the control. After a 30‐day culture, shrimp were infected with V. harveyi and monitored for 10 days. A significant reduction (p < .05) in cumulative mortality was observed in FB‐ and MB‐supplemented shrimp (43.24% and 45.05%, respectively), compared to the control (63.06%). This finding demonstrated that administration of microencapsulated probiotics was as effective as freeze‐dried probiotics for improving growth, feed efficiency, survival, Bacillus in gastrointestinal tracts, water quality (ammonia and nitrite) and conferring disease resistance to V. harveyi.     

First report of isolation and complete genome of Vibrio rotiferianus strain SSVR1601 from cage‐cultured black rockfish (Sebastes schlegelii) associated with skin ulcer

Vibrio rotiferianus is an important marine pathogen of various aquatic organisms and can be found widely distributed in the marine environment. To further characterize this pathogen, the pathogenic properties and genome of V. rotiferianus SSVR1601 isolated from Sebastes schlegelii with skin ulcer were analysed. SSVR1601 was shown to be short rod‐shaped cell with a single polar flagellum. Different degrees of pathological changes in fish kidney, intestine, gills and liver were observed after SSVR1601 challenge. The SSVR1601 genome consists of two chromosomes and two plasmids with a total of 5,717,113 bp, 42.04%–44.93% GC content, 5,269 predicted CDSs, 134 tRNAs and 40 rRNAs. The common virulence factors including OMPs, haemolysin, flagellin, DNase, entF, algU, tcpI, acfB and rfaD were found in strain SSVR1601. Furthermore, factors responsible for iron uptake (fur, fepC and ccmC) and types II, IV and VI secretion systems were detected, which are likely responsible for the pathogenicity of SSVR1601. The antimicrobial resistance genes, bacA, tet34 and norM, were detected based on Antibiotic Resistance Genes Database. The phylogenetic analysis revealed SSVR1601 to be most closely related to V. rotiferianus strains CAIM577 and B64D1.     

The high prevalence of pathogenic Vibrio harveyi with multiple antibiotic resistance in scale drop and muscle necrosis disease of the hybrid grouper,Epinephelus fuscoguttatus (♀) × E. lanceolatus (♂), in China

Scale drop and muscle necrosis disease with high mortality widely occurred recently in the hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), a crucial cultured marine fish species in China. In this study, 30 Harveyi clade isolates of 27 Vibrio harveyi strains were isolated from diseased hybrid groupers in the south‐east and north‐east coastal areas of China. A total of 22 V. harveyi strains were determined to be pathogenic, and most challenged fish died within 2 days of infection; surviving individuals exhibited scale drop and deep dermal lesions as naturally diseased fish. Although five typical virulence genes, including luxR, toxR_Vh, chiA, serine protease and vhh widely existed in V. harveyi, no obvious correlation was established between virulent strains and virulence genes harboured in them. Furthermore, multiple antibiotic resistance was widely exhibited in Harveyi clade strains, particularly for penicillins, polypeptides, lincomycins, acetylspiramycin, streptomycin, metronidazole and bacitracin. And the multiple antibiotic resistance indices were gradually decreased from southern to northern areas of China. This study demonstrated that the pathogenic V. harveyi with multiple antibiotic resistance is highly prevalent in hybrid grouper in China, which requires particular attention.     

The hidden companion of non‐native fishes in north‐east Atlantic waters

A tropicalization phenomenon of ichthyofauna has been described in the last decades in Galicia (north‐eastern Atlantic), with increasing reports of tropical and subtropical fishes appearing northward this distribution range. A search for parasites was carried out in the digestive tract of two specimens first captured in Galician waters: the prickly puffer Ephippion guttifer (Tetraodontidae) and the African stripped grunt Parapristipoma octolineatum (Haemulidae). Examination of E. guttifer showed high intensity of nematodes, from three different genera: Cucullanus (Cucullanidae), Hysterothylacium (Raphidascaridae) and Anisakis (Anisakidae), with demonstrated pathogenicity to humans. Molecular identification allowed the identification of Anisakis pegreffii, already described in the area, and first reports for European waters of Cucullanus dodsworthi, Hysterothylacium reliquens and a new Hysterothylacium sp. P. octolineatum showed a far lower level of parasitization, with two Hysterothylacium larvae, genetically identified as Hysterothylacium deardorffoverstreetorum, also its first report in the eastern Atlantic. Thus, possible ecological impact of the occurrence of two non‐native individual fishes in a new area could be remarkably higher if we see this issue through the lens of the parasitological perspective, as far as only two individual fish can harbour more of one hundred nematode parasites belonging to different species, most of them also new species for that area.     

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.     

Effects of a probiotic mixture (Bacillus subtilis YB‐1 and Bacillus cereus YB‐2) on disease resistance and non‐specific immunity of sea cucumber,Apostichopus japonicus (Selenka)

The effect of a commercially available compound probiotics product containing Bacillus subtilis YB‐1 (50%) and Bacillus cereus YB‐2 (50%) fed to sea cucumbers, Apostichopus japonicus (Selenka) on challenge infections and non‐specific immune responses was assessed. Sea cucumbers (were randomly allocated into nine aquariums at a density of 30 sea cucumbers per tank and triplicate groups) were fed diets containing 0 (control), 10⁷ and 10¹⁰ cfu (g diet)^?1 of the probiotics mixture for 32 days. The growth factors and immunological parameters were measured. In addition, the effects on resistance against Vibrio alginolyticus infection were also evaluated. The results indicate that all the immunological parameters (phagocytic activity, superoxide anion production, lysozyme activity, catalase activity and phenoloxidase activity) measured and the growth rate of sea cucumbers fed 10¹⁰ cfu of the probiotics mixture were significantly (P < 0.05) improved than control groups at 16 and 32 days. After challenging, the cumulative mortality for the control was 100%, whereas the cumulative mortality for sea cucumbers fed 10¹⁰ cfu of the probiotics mixture was 47% (P < 0.05). Although the total autochthonous intestinal heterotrophic bacterial counts were not affected by dietary treatment (P > 0.05), Bacillus sp. levels were significantly elevated in sea cucumbers fed the probiotics mixture (P < 0.05). These results confirmed that administration of the probiotics mixture in the diet stimulated non‐specific immune responses and enhanced the growth performance of sea cucumbers, and was effective in controlling infections caused by V. alginolyticus.     

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.     

Immune responses of Litopenaeus vannamei to non‐ionic ammonia stress: a comparative study on shrimps in freshwater and seawater conditions

To investigate the effect of non‐ionic ammonia (NH₃‐N) stress (0.1 and 0.5 mg L^?1) on the immunity of Litopenaeus vannamei cultured in long‐term freshwater, the total haemocyte count (THC), the activity of phenoloxidase (PO), nitric oxide synthase (NOS), superoxidase dismutase (SOD) and the content of malondialdehyde (MDA) were determined and further compared with those of seawater shrimps. The results showed that NH₃‐N stress significantly reduced THC and the activity of PO and SOD (P < 0.05). Under 0.1 mg L^?1 NH₃‐N stress, NOS activity increased first and then decreased significantly, while it dropped dramatically under 0.5 mg L^?1 NH₃‐N stress (P < 0.05). During NH₃‐N stress, MDA content increased continuously, and the MDA content in hepatopancreas of freshwater shrimps was higher than that of seawater shrimps. It was concluded that NH₃‐N stress significantly influenced the non‐specific immunity and could also upset the balance of antioxidant system of L. vannamei in both freshwater and seawater shrimps. Compared with in seawater, the shrimps in freshwater were more vulnerable to NH₃‐N stress because of higher lipid peroxidation and lower immunity.