Temporal and Spatial Dynamics of Vibrio harveyi: An Environmental Parameter Correlation Investigation in a 4-Metre-Deep Dicentrarchus labrax Aquaculture Tank.

Nowadays, European seabass (Dicentrarchus labrax) aquaculture is undergoing a significant expansion. Nevertheless, the aquaculture industry is plagued by vibriosis. The spatial and temporal dynamics of Vibrio harveyi were studied on a European seabass farm in northern France during seven months of 2022. Concrete specimens were suspended and water was pumped from different depths (0.3 m, 2.15 m and 4 m deep), providing insights into the biofilm and planktonic V. harveyi dynamics. The abundances of V. harveyi, in the biofilm and free-living forms, were positively correlated. The water parameters revealed seasonal fluctuations in temperature, pH, and salinity, with no significant differences observed across the water column. Quantification of V. harveyi revealed no significant differences between depths, but seasonality, with peak abundances observed in August, correlated with temperature increases. Principal component analysis identified temperature as a primary driver, but also additional parameters, such as salinity and pH. Vibriosis occurred during the sampling period, providing valuable insights into the conditions before, during, and after the outbreaks. This study underscores the importance of understanding V. harveyi behaviour in aquaculture, particularly in the context of global warming, for effective disease management and sustainable practices.

Virulent properties and genomic diversity of Vibrio vulnificus isolated from environment, human, diseased fish.

The incidence of Vibrio vulnificus infections, with high mortality rates in humans and aquatic animals, has escalated, highlighting a significant public health challenge. Currently, reliable markers to identify strains with high virulence potential are lacking, and the understanding of evolutionary drivers behind the emergence of pathogenic strains is limited. In this study, we analyzed the distribution of virulent genotypes and phenotypes to discern the infectious potential of V. vulnificus strains isolated from three distinct sources. Most isolates, traditionally classified as biotype 1, possessed the virulence-correlated gene-C type. Environmental isolates predominantly exhibited YJ-like alleles, while clinical and diseased fish isolates were significantly associated with the nanA gene and pathogenicity region XII. Hemolytic activity was primarily observed in the culture supernatants of clinical and diseased fish isolates. Genetic relationships, as determined by multiple-locus variable-number tandem repeat analysis, suggested that strains originating from the same source tended to cluster together. However, multilocus sequence typing revealed considerable genetic diversity across clusters and sources. A phylogenetic analysis using single nucleotide polymorphisms of diseased fish strains alongside publicly available genomes demonstrated a high degree of evolutionary relatedness within and across different isolation sources. Notably, our findings reveal no direct correlation between phylogenetic patterns, isolation sources, and virulence capabilities. This underscores the necessity for proactive risk management strategies to address pathogenic V. vulnificus strains emerging from environmental reservoirs.IMPORTANCEAs the global incidence of Vibrio vulnificus infections rises, impacting human health and marine aquacultures, understanding the pathogenicity of environmental strains remains critical yet underexplored. This study addresses this gap by evaluating the virulence potential and genetic relatedness of V. vulnificus strains, focusing on environmental origins. We conduct an extensive genotypic analysis and phenotypic assessment, including virulence testing in a wax moth model. Our findings aim to uncover genetic and evolutionary factors that drive pathogenic strain emergence in the environment. This research advances our ability to identify reliable virulence markers and understand the distribution of pathogenic strains, offering significant insights for public health and environmental risk management.

Antibiotic resistance trends among Vibrio vulnificus and Vibrio parahaemolyticus isolated from the Chesapeake Bay, Maryland: a longitudinal study.

Antibiotics are often used to treat severe Vibrio infections, with third-generation cephalosporins and tetracyclines combined or fluoroquinolones alone being recommended by the US Centers for Disease Control and Prevention. Increases in antibiotic resistance of both environmental and clinical vibrios are of concern; however, limited longitudinal data have been generated among environmental isolates to inform how resistance patterns may be changing over time. Hence, we evaluated long-term trends in antibiotic resistance of vibrios isolated from Chesapeake Bay waters (Maryland) across two 3-year sampling periods (2009-2012 and 2019-2022). Vibrio parahaemolyticus (n = 134) and Vibrio vulnificus (n = 94) toxR-confirmed isolates were randomly selected from both sampling periods and tested for antimicrobial susceptibility against eight antibiotics using the Kirby-Bauer disk diffusion method. A high percentage (94%-96%) of V. parahaemolyticus isolates from both sampling periods were resistant to ampicillin and only 2%-6% of these isolates expressed intermediate resistance or resistance to third-generation cephalosporins, amikacin, tetracycline, and trimethoprim-sulfamethoxazole. Even lower percentages of resistant V. vulnificus isolates were observed and those were mostly recovered from 2009 to 2012, however, the presence of multiple virulence factors was observed. The frequency of multi-drug resistance was relatively low (6%-8%) but included resistance against antibiotics used to treat severe vibriosis in adults and children. All isolates were susceptible to ciprofloxacin, a fluoroquinolone, indicating its sustained efficacy as a first-line agent in the treatment of severe vibriosis. Overall, our data indicate that antibiotic resistance patterns among V. parahaemolyticus and V. vulnificus recovered from the lower Chesapeake Bay have remained relatively stable since 2009.IMPORTANCEVibrio spp. have historically been susceptible to most clinically relevant antibiotics; however, resistance and intermediate-resistance have been increasingly recorded in both environmental and clinical isolates. Our data showed that while the percentage of multi-drug resistance and resistance to antibiotics was relatively low and stable across time, some Vibrio isolates displayed resistance and intermediate resistance to antibiotics typically used to treat severe vibriosis (e.g., third-generation cephalosporins, tetracyclines, sulfamethoxazole-trimethoprim, and aminoglycosides). Also, given the high case fatality rates observed with Vibrio vulnificus infections, the presence of multiple virulence factors in the tested isolates is concerning. Nevertheless, the continued susceptibility of all tested isolates against ciprofloxacin, a fluoroquinolone, is indicative of its use as an effective first-line treatment of severe Vibrio spp. infections stemming from exposure to Chesapeake Bay waters or contaminated seafood ingestion.

A novel vaccination strategy against Vibrio harveyi infection in Asian seabass (Lates calcarifer) with the aid of oxygen nanobubbles and chitosan.

Immersion vaccination, albeit easier to administer than immunization by injection, sometimes has challenges with antigen uptake, resulting in sub-optimal protection. In this research, a new strategy to enhance antigen uptake of a heat-inactivated Vibrio harveyi vaccine in Asian seabass (Lates calcarifer) using oxygen nanobubble-enriched water (ONB) and positively charged chitosan (CS) was explored. Antigen uptake in fish gills was assessed, as was the antibody response and vaccine efficacy of four different combinations of vaccine with ONB and CS, and two control groups. Pre-mixing of ONB and CS before introducing the vaccine, referred to as (ONB + CS) + Vac, resulted in superior antigen uptake and anti-V. harveyi antibody (IgM) production in both serum and mucus compared to other formulas. The integration of an oral booster (4.22 × 108 CFU/g, at day 21-25) within a vaccine trial experiment set out to further evaluate how survival rates post exposure to V. harveyi might be improved. Antibody responses were measured over 42 days, and vaccine efficacy was assessed through an experimental challenge with V. harveyi. The expression of immune-related genes IL1ß, TNFα, CD4, CD8, IgT and antibody levels were assessed at 1, 3, and 7-day(s) post challenge (dpc). The results revealed that antibody levels in the group (ONB + CS) + Vac were consistently higher than the other groups post immersion immunization and oral booster, along with elevated expression of immune-related genes after challenge with V. harveyi. Ultimately, this group demonstrated a significantly higher relative percent survival (RPS) of 63 % ± 10.5 %, showcasing the potential of the ONB-CS-Vac complex as a promising immersion vaccination strategy for enhancing antigen uptake, stimulating immunological responses, and improving survival of Asian seabass against vibriosis.

Compounds isolation from Syzygium cumini leaf extract against the Vibrio species in shrimp through bioassay-guided fractionation.

This study was conducted to isolate and identify the bioactive compounds from the ethanolic extract of Syzygium cumini leaf against Vibrio species through a bioassay-guided fractionation. The ethanol extract was exposed to silica gel chromatography followed by reversed phase HPLC to isolate the most effective fraction against V. parahaemolyticus. Using further UHPLC-orbitrap-ion trap mass spectrometry, five compounds were isolated with broad-spectrum potency against a range of Vibrio species viz. V. parahaemolyticus, V. alginolyticus, V. harveyi, V. vulnificus and V. anguillarum. The IC50 values for the compounds ranged from 8 to 48 µg/mL against the most sensitive species V. vulnificus and 58 to >400 µg/mL against V. alginolyticus. The results of the toxicity tests demonstrated that the compounds were not harmful for shrimp. The study's findings indicate that S. cumini leaf extract may contain bioactive molecules that are able to be substituted for antibiotics to treat vibriosis in shrimp farming.

Anti-vibriosis bioactive molecules from marine-derived variant Streptomyces sp. ZZ741A.

The infection of vibrio is an important cause of huge economic losses in aquaculture industry. At present, antibiotics are mainly used to prevent and reduce the infection of the vibrio, which has accelerated the emergence of multi-drug-resistant strains. New generation alternative anti-vibrio drugs were in urgent to solve this problem. In this study, six compounds (1-6) were isolated from the Streptomyces sp. ZZ741A, a marine-derived Streptomyces variant, including one new compound, 2-carbamoylphenyl isobutyrate (1), five known ones, nocardamine (2), dehydroxynocardamine (3), phenylacetic acid (4), thiophenol (5) and 2,3-dihydroxybenzoic acid (6). The anti-vibriosis assay showed that compounds 2 and 3 had specific inhibition activity against Vibrio vulnificus, Vibrio alginolyticus, and Vibrio parahaemolyticus with the MIC values ranging from 8 to 128 µg/mL. The molecular docking study of their possible mechanism of anti-vibriosis activity showed that the activity might come from the inhibition of Outer membrane protein U (OmpU).

Immuno-protective response of Asian seabass (Lates calcarifer) to inactivated vaccines against Streptococcus iniae and Vibrio harveyi.

BACKGROUND: In this study, the protective immunity and immunogenicity of the monovalent and bivalent Streptococcus iniae and Vibrio harveyi vaccine were evaluated in Asian seabass. To analyze immune responses, 1200 Asian seabass with an average weight of 132.6 ± 25.4 g were divided into eight treatments in triplicates (50 fish per tank) as follows: S. iniae immunized by injection (SI), V. harveyi immunized by injection (VI), bivalent S. iniae and V. harveyi (SVI) immunized by injection, S. iniae immunized by immersion (SIM), V. harveyi (VIM) immunized by immersion, bivalent S. iniae and V. harvei (SVIM) immunized by immersion, phosphate-buffered saline (PBS) by injection (PBSI) and control group without vaccine administration (CTRL). Blood and serum samples were taken at the end of the 30th and 60th days. Then the vaccinated groups were challenged with two bacteria (S. iniae) and (V. harveyi) separately and mortality was recorded for 14 days. RESULTS: This study reveals that there is no significant difference in the hematological parameters on the 30th and 60th days of the experiment in the vaccine-immunized groups compared to the CTRL group (P > 0.05). Meanwhile, there was no significant difference in the amount of serum albumin level, respiratory burst activity, and serum bactericidal activity in the vaccine-immunized groups compared to the CTRL group on the 30th and 60th days of the experiment (P > 0.05). Total protein on the 60th day (in the VI and SVI groups), globulin on the 30th day (in the VI and SVI groups) and the 60th day (in the VI group) compared to the CTRL and PBSI groups had a significant increase (P < 0.05). Complement activity (in the VI and SVI groups) and lysozyme (in the SI and SVI groups) increased significantly compared to the control group (P < 0.05). Serum antibody titer against S. iniae had a significant increase in the SI, VI, SVI and SVIM groups compared to the CTRL and PBSI groups (P < 0.05). Serum antibody titer against V. harveyi had a significant increase in the groups immunized with the vaccine compared to the CTRL and PBSI groups (P < 0.05). A significant increase in the relative percentage survival (RPS) following challenge with S. iniae in the SVI (86.6%), SI (83.3%,) and VI (73.3%) groups were observed compared to the CTRL (43.3%) and PBSI (40%) groups (P < 0.05). Also, a significant increase in the RPS after challenge with V. harveyi in the SVI group, VI 86.6%, SVI 83.3%, VIM 80% and SVIM 76.6% were observed compared to the CTRL (46.6%) and PBSI (50%) groups (P < 0.05). CONCLUSION: Overall, the results demonstrated that the bivalent vaccine of S. iniae and V. harveywas able to produce significant immunogenicity and RPS in Asian seabass.

Morinda citrifolia fruit extract enhances the resistance of Penaeus vannamei to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus is a gram-negative facultative anaerobic bacterium implicated as the causative agent of several shrimp diseases. As part of the effort to provide biocontrol and cost-effective treatments, this research was designed to elucidate the effect of Morinda citrifolia fruit extract on the immunity of Penaeus vannamei postlarvae (PL) to V. parahaemolyticus. The methanol extract of M. citrifolia was vacuum evaporated, and the bioactive compounds were detected using gas chromatography‒mass spectrometry (GC‒MS). Thereafter, P. vannamei PL diets were supplemented with M. citrifolia at different concentrations (0, 10, 20, 30, 40, and 50 mg/g) and administered for 30 days before 24 h of exposure to the bacterium V. parahaemolyticus. A total of 45 bioactive compounds were detected in the methanol extract of M. citrifolia, with cyclononasiloxane and octadecamethyl being the most abundant. The survival of P. vannamei PLs fed the extract supplement was better than that of the control group (7.1-26.7% survival greater than that of the control group) following V. parahaemolyticus infection. Shrimp fed 50 mg/g M. citrifolia had the highest recorded survival. The activities of digestive and antioxidant enzymes as well as hepatopancreatic cells were significantly reduced, except for those of lipase and hepatopancreatic E-cells, which increased following challenge with V. parahaemolyticus. Histological assessment of the hepatopancreas cells revealed reduced cell degeneration following the administration of the plant extracts (expecially those fed 50 mg/g M. citrifolia) compared to that in the control group. Therefore, the enhanced immunity against V. parahaemolyticus infection in P. vannamei could be associated with the improved hepatopancreas health associated with M. citrifolia fruit extract supplementation.

Comparative proteome analysis revealed potential biomarkers and the underlying immune mechanisms in Vibrio-resistant hybrid grouper, Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂.

Vibrio alginolyticus is the causative agent of vibriosis, a common bacterial infection in grouper aquaculture that is associated with the development of haemorrhagic and non-haemorrhagic ulcerations on the fish. In the present study, comparative proteome analysis was performed on serum samples from Vibrio-resistant and Vibrio-susceptible grouper. Samples were analysed using high-throughput LC-MS/MS and identified 2770 unique peptides that corresponded to 344 proteins. Subsequent analysis identified 21 proteins that were significantly up-regulated in the resistant group compared to the control and the susceptible groups. Those proteins are associated with immunostimulatory effects, signalling and binding cascade, metabolism, and maintaining tissue integrity and physiological condition. Besides, potential protein biomarkers related to the immune system were identified, which could be associated with the disease-resistant phenotype. These data provide insights into the underlying immune mechanism of hybrid groupers upon Vibrio sp. infection.

Draft genome sequence of Bacillus velezensis strain 3TSA-3, a potential probiotic for Pacific white shrimp Penaeus vannamei postlarvae isolated from commercial hatchery tanks.

We report the draft genome of Bacillus velezensis strain 3TSA-3, isolated from Pacific white shrimp Penaeus vannamei postlarvae collected from a hatchery tank with high survival despite the presence of pathogenic Vibrio. The strain possesses genes encoding bacteriocins and lacks virulence factor genes, characteristics for a potential aquaculture probiotic.

Positive cases of bovine genital campylobacteriosis and bovine trichomonosis in the eradication program of La Pampa, Argentina: 2008-2021.

Bovine genital campylobacteriosis, caused by the gram-negative bacteria Campylobacter fetus venerealis, and bovine trichomonosis, caused by the parasite protozoan Tritrichomonas foetus, are venereal diseases that occur with long intercalving periods and abortion. The control of both diseases relies on microbiological testing and culling infected bulls. Vaccination and antibiotic treatment may help in controlling campylobacteriosis but are not recommended for trichomonosis control. Several regions of the world have active control programs for trichomonosis, not campylobacteriosis. In Argentina, the state of La Pampa aims to eradicate trichomonosis and campylobacteriosis by imposing annual diagnostic testing of every bull and slaughtering positive animals. Prior studies indicated a declining trend in the prevalence of campylobacteriosis and trichomonosis in La Pampa. It was also proposed that the prevalence of one disease could be estimated from the prevalence of the other. The purpose of this retrospective analysis of data gathered from 2008 to 2021 was to determine the La Pampa program's efficacy. Descriptive statistics were employed to determine the reason behind the correlation between tricomonosis and campylobacteriosis diagnostic results. The outcomes refute the notion that this program of venereal eradication was a success. Furthermore, an excess of false positives in both diagnoses may have contributed to the correlation between the prevalences of campylobactriosis and trichomonosis. The practice of killing animals without verifying positive results hinders the determination of disease prevalence and results in the death of numerous healthy animals.

Detection of non-pathogenic and pathogenic populations of Vibrio parahaemolyticus in various samples by the conventional, quantitative and droplet digital PCRs.

In this study, three generations of polymerase chain reaction (PCR) assays: (i) conventional PCR, (ii) qPCR and (iii) droplet digital PCR (ddPCR), were systematically tested for their abilities to detect non-pathogenic and pathogenic populations of Vibrio parahaemolyticus. The limit of detection (LOD) for the ddPCR was 1.1 pg/µL of purified DNA, followed by the qPCR (5.6 pg/µL) and the conventional PCR (8.8 pg/µL). Regarding the LOD for V. parahaemolyticus cells, the ddPCR assay was able to detect 29 cells, followed by the conventional PCR assay (58 cells) and the qPCR assay (115 cells). Regarding the sensitivities to detect this pathogen from PCR inhibition prone samples (naturally contaminated mussels), the ddPCR assay significantly outperformed the conventional PCR and qPCR. The ddPCR assay was able to consistently detect non-pathogenic and pathogenic populations of V. parahaemolyticus from naturally contaminated mussels, indicating its tolerance to various PCR inhibitors. This study also revealed the significant difference between conventional PCR and qPCR. The conventional PCR assay showed significantly greater sensitivity than that of the qPCR assay in detecting V. parahaemolyticus in crude samples, whereas the qPCR assay showed better sensitivity in detecting the presence of V. parahaemolyticus in purified DNA samples.

Marine actinomycete Streptomyces variabilis S26 as a biocontrol agent for vibriosis in shrimp larval rearing systems.

Indiscriminate use of antibiotics has led to the emergence of antibiotic-resistant microbes and the loss of natural flora in aquaculture systems necessitating the ban of many of these chemotherapeutants in aquaculture. Actinobacteria play a profound role in the biogeochemical cycling in the marine environment and represent the principal source of secondary metabolites with antimicrobial property. In the present study, 98 marine-derived actinomycete isolates were screened for antimicrobial activity against the common aquatic pathogens. A potent actinomycete isolate S26, identified as Streptomyces variabilis based on 16 S ribosomal RNA (rRNA) gene sequencing was then checked for the production of antibiotic in five different fermentation media and the one which showed maximum production was chosen for further study. Optimization of the fermentation medium for secondary metabolite production was carried out by response surface methodology (RSM) using DESIGN EXPERT. The analysis of variance (ANOVA) of the quadratic regression model demonstrated that the model was highly significant for the response concerned that is, antimicrobial activity as evident from the Fisher's F- test with a very low probability value [(P model>F) = 0.0001]. Of the 10 different solutions suggested by the software, the most suitable composition was found to be starch, 1.38%; soy powder, 0.88%; ammonium sulfate, 0.16% and salinity, 27.76‰. S. variabilis S26 cultured in the optimized production medium was applied in the Penaeus monodon larval rearing system and the total Vibrio count and survival rate were estimated. S. variabilis S26 treatment showed a significant reduction in vibrios and conferred better protection to P. monodon in culture system compared with control.

In silico designing and characterization of outer membrane protein K (OmpK) from Vibrio anguillarum and its expression in Nicotiana tabacum for the development of a plant-based vaccine against fish vibriosis.

Vibriosis is caused by Vibrio anguillarum in various species of aquaculture. A novel, secure, and stable vaccine is needed to eradicate vibriosis. Here, for reverse vaccinology and plant-based expression, the outer membrane protein K (OmpK) of V. anguillarum was chosen due to its conserved nature in all Vibrio species. OmpK, an ideal vaccine candidate against vibriosis, demonstrated immunogenic, non-allergic, and non-toxic behavior by using various bioinformatics tools. Docking showed the interaction of the OmpK model with TLR-5. In comparison to costly platforms, plants can be used as alternative and economic bio-factories to produce vaccine antigens. We expressed OmpK antigen in Nicotiana tabacum using Agrobacterium-mediated transformation. The expression vector was constructed using Gateway® cloning. Transgene integration was verified by polymerase chain reaction (PCR), and the copy number via qRT-PCR, which showed two copies of transgenes. Western blotting detected monomeric form of OmpK protein. The total soluble protein (TSP) fraction of OmpK was equivalent to 0.38% as detected by ELISA. Mice and fish were immunized with plant-derived OmpK antigen, which showed a significantly high level of anti-OmpK antibodies. The present study is the first report of OmpK antigen expression in higher plants for the potential use as vaccine in aquaculture against vibriosis, which could provide protection against multiple Vibrio species due to the conserved nature OmpK antigen.

Increased incidence of vibriosis in Maryland, U.S.A., 2006-2019.

BACKGROUND: Vibrio spp. naturally occur in warm water with moderate salinity. Infections with non-cholera Vibrio (vibriosis) cause an estimated 80,000 illnesses and 100 fatalities each year in the United States. Climate associated changes to environmental parameters in aquatic ecosystems are largely promoting Vibrio growth, and increased incidence of vibriosis is being reported globally. However, vibriosis trends in the northeastern U.S. (e.g., Maryland) have not been evaluated since 2008. METHODS: Vibriosis case data for Maryland (2006-2019; n = 611) were obtained from the COVIS database. Incidence rates were calculated using U.S. Census Bureau population estimates for Maryland. A logistic regression model, including region, age group, race, gender, occupation, and exposure type, was used to estimate the likelihood of hospitalization. RESULTS: Comparing the 2006-2012 and 2013-2019 periods, there was a 39% (p = 0.01) increase in the average annual incidence rate (per 100,000 population) of vibriosis, with V. vulnificus infections seeing the greatest percentage increase (53%, p = 0.01), followed by V. parahaemolyticus (47%, p = 0.05). The number of hospitalizations increased by 58% (p = 0.01). Since 2010, there were more reported vibriosis cases with a hospital duration ≥10 days. Patients from the upper eastern shore region and those over the age of 65 were more likely (OR = 6.8 and 12.2) to be hospitalized compared to other patients. CONCLUSIONS: Long-term increases in Vibrio infections, notably V. vulnificus wound infections, are occurring in Maryland. This trend, along with increased rates in hospitalizations and average hospital durations, underscore the need to improve public awareness, water monitoring, post-harvest seafood interventions, and environmental forecasting ability.

Mass mortality of the keratose sponge Sarcotragus foetidus in the Aegean Sea (Eastern Mediterranean) correlates with proliferation of Vibrio bacteria in the tissues.

In the last two decades, episodes of mass mortality in benthic communities have often been associated with climatic anomalies, but the ultimate mechanisms through which they lead to death have rarely been identified. This study reports a mass mortality of wild sponges in the Aegean Sea (Turkey, Eastern Mediterranean), which affected the keratose demosponge Sarcotragus foetidus in September 2021. We examined the occurrence of thermo-dependent bacteria of the genus Vibrio in the sponges, identified through 16S rRNA of colonies isolated from sponge tissue in specific culturing media. Six Vibrio sequences were identified from the sponges, three of them being putatively pathogenic (V. fortis, V. owensii, V. gigantis). Importantly, those Vibrios were isolated from only tissues of diseased sponges. In contrast, healthy individuals sampled in both summer and winter led to no Vibrio growth in laboratory cultures. A 50 years record of sea surface temperature (SST) data for the study area reveals a progressive increase in temperature from 1970 to 2021, with values above 24°C from May to September 2021, reaching an absolute historical maximum of 28.9°C in August 2021. We hypothesize that such elevated SST values maintained for several months in 2021 promoted proliferation of pathogenic Vibrio species (thermo-dependent bacteria) in S. foetidus, triggering or aggravating the course of sponge disease. Thus, vibrioisis emerges as one of the putative mechanisms through which global water warming in the Mediterranean Sea translates into sponge mortality. The historical time course of temperature data for the studied area in the Aegean Sea predicts that recurrent waves of elevated SST are likely to occur in the coming summers. If so, recurrent disease may eventually eliminate this abundant sponge from the sublittoral in the midterm, altering the original bathymetric distribution of the species and compromising its ecological role.

Design of a multi-epitope vaccine (vme-VAC/MST-1) against cholera and vibriosis based on reverse vaccinology and immunoinformatics approaches.

Vibriosis and cholera are serious diseases distributed worldwide and caused by six marine bacteria of the Vibrio genus. Thousands of deaths occur each year due to these illnesses, necessitating the development of new preventive measures. Presently, the existing cholera vaccine demonstrates an effectiveness of approximately 60%. Here we describe a new multi-epitope vaccine, 'vme-VAC/MST-1' based on vaccine targets identified by reverse vaccinology and epitopes predicted by immunoinformatics, two currently effective tools for predicting new vaccines for bacterial pathogens. The vaccine was designed to combat vibriosis and cholera by incorporating epitopes predicted for CTL, HTL, and B cells. These epitopes were identified from six vaccine targets revealed through subtractive genomics, combined with reverse vaccinology, and were further filtered using immunoinformatics approaches based on their predicted immunogenicity. To construct the vaccine, 28 epitopes (24 CTL/B and 4 HTL/B) were linked to the sequence of the cholera toxin B subunit adjuvant. In silico analyses indicate that the resulting immunogen is stable, soluble, non-toxic, and non-allergenic. Furthermore, it exhibits no homology to the host and demonstrates a strong capacity to elicit innate, B-cell, and T-cell immune responses. Our analysis suggests that it is likely to elicit immune reactions mediated through the TLR5 pathway, as evidenced by the molecular docking of the vaccine with the receptor, which revealed high affinity and a favorable reaction. Thus, vme-VAC/MST-1 is predicted to be a safe and effective solution against pathogenic Vibrio spp. However, further experimental analyses are required to measure the vaccine's effects In vivo.Communicated by Ramaswamy H. Sarma.

Feeding Strategies with Multi-Strain Probiotics Affect Growth, Health Condition, and Disease Resistance in Asian Seabass (Lates calcarifer).

A 16-week feeding trial was done to examine the impacts of continuous feeding (CF) or pulse-feeding (PF) of multi-strain probiotics on Asian seabass (Lates calcarifer, 30.0 ± 0.1 g) juveniles. In this study, three different multi-strain probiotic mixtures were added to a basal diet, including (I) a mixture of different strains of Lactobacillus plantarum, (II) a mixture of the first probiotic (I) + L. delbrueckii sub bulgaricus, L. rhamnosus and L. acidophilus, and (III) a mixture of the second probiotic (II) + two quorum quenching (QQ) bacteria (Bacillus thuringiensis QQ1 and B. cereus QQ2). CF (every day) or PF (every two weeks) strategies were applied for using the abovementioned probiotics to design seven experimental groups including C (control, without probiotics), CF-I (continuous feeding of fish with the probiotic mixture I), CF-II (continuous feeding of fish with the probiotic mixture II), CF-III (continuous feeding of fish with the probiotic mixture III), PF-I (pulse-feeding of fish with the probiotic mixture I), PF-II (pulse-feeding of fish with the probiotic mixture II), and PF-III (pulse-feeding of fish with the probiotic mixture III). Four hundred and twenty fish were stocked into 21 circular polyethylene tanks with 220 L volume (20 fish/tank). Each dietary treatment had three replicates. Tanks were supplied with seawater (temperature = 30.5 °C, salinity = 45 g L-1) in a flow-throw system. Fish in CF-I, CF-II, and CF-III had higher growth rate (ca. 113-145%) and better feed conversion ratio than fish fed C and PF-I (P < 0.05). Fish in the CF-III group had the highest protease activity. Continuous feeding strategy resulted in a higher amount of glutathione and catalase activities in both the liver and plasma as well as higher superoxide dismutase activity in the liver of fish. Pulse-feeding strategy resulted in lower plasma lactate dehydrogenase and aspartate aminotransferase levels than the CF strategy. Regardless of feeding strategy, different probiotic mixtures significantly enhanced blood hemoglobin and hematocrit levels compared to the control. Continuous feeding with the multi-strain probiotics resulted in a higher survival rate against Vibrio harveyi than the PF method. Continuous feeding induced higher mRNA transcription levels of granulocyte-macrophage colony-forming cells and interleukin 10 genes in the gut of fish than PF strategy. In conclusion, continuous feeding with multi-strain probiotics is better than pulse-feeding on growth, feed utilization, antioxidant capacity, and the gut's immune-related genes and led to higher resistance of L. calcarifer in challenge with V. harveyi.

In Vivo Anti-Vibrio Evaluation of Sponge-Associated Bacteria on the Survival Rate of Litopenaeus vannamei Infected with Pathogenic Vibrio Species.

Sponge-associated bacteria are considered a rich source of bioactive compounds particularly to reduce the risk of Vibrio harveyi and Vibrio parahaemolyticus infection. The present study aimed to analyse the effectiveness of 19 isolates to control Vibrio infection in vivo. All 19 isolates displayed a non-pathogenic characteristic on shrimps (cell density of 106 cells/mL) as analysed using the pathogenicity test. The mortality caused by both Vibrio spp. on 50% of the shrimp population (LC50 value) had a cell density of 105 cells/mL as determined using the proportion interval method. On the basis of the challenge test, all isolates improved the survival rate of infected shrimps in diverse effectivities up to 89%, which was nearly 30% higher than the infected control. Two isolates coded as D6.9, and P5.20 reduced shrimp mortality after infection with Vibrio spp. 16S rRNA-based identification showed these isolates were closely similar to different genera of Bacillus and Staphylococcus. The extract derived from the most prospective isolate, D6.9, was dominated by 1-hydroxy-6-(3-isopropenyl-cycloprop-1-enyl)-6-methyl-heptan-2-one, hexadecanoic acid, 4-epicyclomusalenone [(24S)-24-methyl-28-norcycloart-25-en-3-one], and 2,4-dimethyl acetoacetanilide. This observation suggested these isolates characterised by in vivo anti-Vibrio activity need to be further developed as biocontrol candidates.

Isolation, Identification, and Pathogenicity of Vibrio gigantis Retrieved from European Seabass (Dicentrarchus labrax) Farmed in Türkiye.

In this study, V. gigantis strain C24 was isolated from cases of winter mortalities of hatchery-reared European seabass (Dicentrarchus labrax) broodstock in Türkiye. The first mortalities were reported in September 2016 and occurred annually in early autumn/late winter until the end of February 2019, when 15% of accumulated mortality was recorded. Diseased moribund fish exhibited general septicemic signs, including dermal ulcerations with hemorrhagic margins, distended abdomens, and hemorrhages below the pectorals, pelvic fins, and at the operculum. Postmortem findings showed congestion in several internal organs, hemorrhagic ascitic fluid, and congested prolapsed anal openings. The representative bacterial isolate V. gigantis strain C24 was characterized as Gram-negative, motile, nitrite-producing, and as vibrio static agent O/129-sensitive. The full-length 16S rRNA sequence (Accession No. ON778781) and gyrB gene sequence (Accession No. ON792326) of the C24 strain showed high similarity to V. gigantis strains. Moreover, the whole-genome average nucleotide identity (ANI) values (ANI > 97.7%) against four V. gigantis strains above the species demarcation limit unambiguously identified the C24 isolate as a member of this species. A preliminary virulence-gene analysis showed that the V. gigantis isolate C24 encoded at least three exotoxins, including two aerolysins and a thermolabile hemolysin. The experimental infection showed that the C24 isolate exhibited low to moderate virulence in experimentally infected European seabass juveniles. Interestingly, antimicrobial susceptibility testing revealed that the C24 isolate was susceptible to nalidixic acid, ciprofloxacin, and several other antibiotics but resistant to tilmicosin, kanamycin, streptomycin, and ampicillin. To our knowledge, this study is the first to report that V. gigantis could be considered an emerging bacterial pathogen in Türkiye, and it may threaten the international European seabass production.