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.

Potential therapeutic use of dermaseptin S4 from the frog Phyllomedusa sauvagii and its derivatives against bacterial pathogens in fish.

AIM: Dermaseptins are one of the main families of antimicrobial peptides (AMPs) derived from the skin secretions of Hylidae frogs. Among them, dermaseptin S4 (DS4) is characterized by its broad-spectrum of activity against bacteria, protozoa, and fungi. In this study, the physicochemical properties of the native peptide DS4 (1-28) and two derivatives [DS4 (1-28)a and DS4 (1-26)a] isolated from the skin of the frog Phyllomedusa sauvagii were investigated and their antimicrobial properties against two marine pathogenic bacteria (Vibrio harveyi and Vibrio anguillarum) were examined. METHODS AND RESULTS: The results indicate that the peptide DS4 (1-26)a has high-antibacterial activity against the tested strains and low-hemolytic activity (<30% lysis at the highest tested concentration of 100 µg/mL) compared to the other two peptides tested. In addition, all three peptides affect the membrane and cell wall integrity of both pathogenic bacteria, causing leakage of cell contents, with DS4 (1-26)a having the most severe impact. These skills were corroborated by transmission electron microscopy and by the variation of cations in their binding sites due to the effects caused by the AMPs. CONCLUSIONS: These results suggest that DS4 and its derivatives, in particular the truncated and amidated peptide DS4 (1-26)a could be effective in the treatment of infections caused by these marine pathogenic bacteria. Future studies are required to validate the use of DS4  in vivo for the prevention of bacterial diseases in fish.

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.

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.

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.

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.

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.

Occurrence of virulence determinants in vibrio cholerae, vibrio mimicus, vibrio alginolyticus, and vibrio parahaemolyticus isolates from important water resources of Eastern Cape, South Africa.

BACKGROUND: Virulence determinants are crucial to the risk assessment of pathogens in an environment. This study investigated the presence of eleven key virulence-associated genes in Vibrio cholerae (n = 111) and Vibrio mimicus (n = 22) and eight virulence determinants in Vibrio alginolyticus (n = 65) and Vibrio parahaemolyticus (n = 17) isolated from six important water resources in Eastern Cape, South Africa, using PCR techniques. The multiple virulence gene indexes (MVGI) for sampling sites and isolates as well as hotspots for potential vibriosis outbreaks among sampling sites were determined statistically based on the comparison of MVGI. RESULT: The PCR assay showed that all the V. cholerae isolates belong to non-O1/non-O139 serogroups. Of the isolates, Vibrio Cholera (84%), V. mimicus (73%), V. alginolyticus (91%) and V. parahaemolyticus (100%) isolates harboured at least one of the virulence-associated genes investigated. The virulence gene combinations detected in isolates varied at sampling site and across sites. Typical virulence-associated determinants of V. cholerae were detected in V. mimicus while that of V. parahaemolyticus were detected in V. alginolyticus. The isolates with the highest MVGI were recovered from three estuaries (Sunday river, Swartkopps river, buffalo river) and a freshwater resource (Lashinton river). The cumulative MVGI for V. cholerae, V. mimicus, V. alginolyticus and V. parahaemolyticus isolates were 0.34, 0.20, 0.45, and 0.40 respectively. The targeted Vibrio spp. in increasing order of the public health risk posed in our study areas based on the MVGI is V. alginolyticus > V. parahaemolyticus > V. cholerae > V. mimicus. Five (sites SR, PA5, PA6, EL4 and EL6) out of the seventeen sampling sites were detected as the hotspots for potential cholera-like infection and vibriosis outbreaks. CONCLUSIONS: Our findings suggest that humans having contact with water resources in our study areas are exposed to potential public health risks owing to the detection of virulent determinants in human pathogenic Vibrio spp. recovered from the water resources. The study affirms the relevancy of environmental Vibrio species to the epidemiology of vibriosis, cholera and cholera-like infections. Hence we suggest a monitoring program for human pathogenic Vibrio spp. in the environment most especially surface water that humans have contact with regularly.

Genome-wide DNA methylation mediates the resistance to vibriosis in Cynoglossus semilaevis.

Chinese tongue sole (Cynoglossus semilaevis) is an economically important marine fish in China. However, vibriosis has caused huge mortality and economic losses in its culturing industry. To reveal the effect of DNA methylation on the resistance to vibriosis in tongue sole, we conducted RNA sequencing and whole genome bisulfite sequencing (WGBS), and compared the gene expressions and DNA methylation patterns between the resistant and susceptible families. We identified a total of 741 significantly differentially expressed genes (DEGs) in kidney and 17460 differentially methylated genes (DMGs), which were both enriched in immune-related pathways, such as "cAMP signaling pathway" and "NOD-like receptor signaling pathway". Through the correlation analysis of DEGs and DMGs, we identified two important immune pathways, including "complement and coagulation cascades", and "cytokine-cytokine receptor interaction", which played important roles in regulating the inflammation level and immune homeostasis. For example, the expression of proinflammatory cytokine il17c was down-regulated under the regulation of DNA methylation; in addition, the expression of protease-activated receptor 3 (par3) was up-regulated, which could induce the up-expressionof il8. These results demonstrated that the regulation of DNA methylation on the genes involved in immune responses might contribute to the resistance to vibriosis in tongue sole, and provided a basis for the control of diseases in fish aquaculture.

Hepcidin and piscidin modulation and antibacterial response in gilthead seabream (Sparus aurata) infected with Vibrio harveyi.

Vibriosis is an infectious disease that generates large economic losses in Mediterranean aquaculture. Vibrio harveyi is one of the marine bacteria causing this disease, it is widespread in the Mediterranean Sea and causes ulcers on the skin of the fish it infects. In addition, the skin is a route of entry and colonization of this pathogen. In this study, one group of fish was injected intraperitoneally with phosphate buffered saline (control group) and another with V. harveyi (infected group). At 4 h after injection, samples of skin mucus, blood, skin, head kidney, liver, and spleen were collected to study the immune response generated. Liver histology showed notable alterations in hepatocyte morphology, such as increased vacuolization. Bactericidal activity was measured in skin mucus and serum against V. harveyi and V. anguillarum, different changes in this activity were recorded depending on the bacteria target and sample (skin mucus or serum) used. Gene expression of genes encoding hepcidins and piscidins (antimicrobial peptides) was performed in the mentioned organs. The results indicated a different expression according to the type of AMP and the tissue studied. Hepcidin appeared involved in all tissues studied while piscidins were in the spleen. In this study we have integrated hepcidin-piscidin modulation with the effects of infection on skin mucosa, serum and hepatocyte morphology. Knowing the changes produced in all these parameters improves the understanding of the infection in the first hours in sea bream and could have applications in the diagnosis or treatment of vibriosis in fish farms.

Evidence that fish death after Vibrio vulnificus infection is due to an acute inflammatory response triggered by a toxin of the MARTX family.

Vibrio vulnificus is an emerging zoonotic pathogen associated with fish farms that is capable of causing a hemorrhagic septicemia known as warm-water vibriosis. According to a recent transcriptomic and functional study, the death of fish due to vibriosis is more related to the inflammatory response of the host than to the tissue lesions caused by the pathogen. In this work, we hypothesize that the RtxA1 toxin (a V. vulnificus toxin of the MARTX (Multifunctional Autoprocessing Repeats in Toxin) family) is the key virulence factor that would directly or indirectly trigger this fatal inflammatory response. Our hypothesis was based on previous studies that showed that rtxA1-deficient mutants maintained their ability to colonize and invade, but were unable to kill fish. To demonstrate this hypothesis, we infected eels (model of fish vibriosis) by immersion with a mutant deficient in RtxA1 production and analyzed their transcriptome in blood, red blood cells and white blood cells during early vibriosis (0, 3 and 12 h post-infection). The transcriptomic results were compared with those obtained in the previous study in which eels were infected with the V. vulnificus parental strain, and were functionally validated. Overall, our results confirm that fish death after V. vulnificus infection is due to an acute, early and atypical inflammatory response triggered by RtxA1 in which red blood cells seem to play a central role. These results could be relevant to other vibriosis as the toxins of this family are widespread in the Vibrio genus.

Genomic analysis and biological characterization of a novel Schitoviridae phage infecting Vibrio alginolyticus.

Vibrio alginolyticus is a Gram-negative bacterium commonly associated with mackerel poisoning. A bacteriophage that specifically targets and lyses this bacterium could be employed as a biocontrol agent for treating the bacterial infection or improving the shelf-life of mackerel products. However, only a few well-characterized V. alginolyticus phages have been reported in the literature. In this study, a novel lytic phage, named ΦImVa-1, specifically infecting V. alginolyticus strain ATCC 17749, was isolated from Indian mackerel. The phage has a short latent period of 15 min and a burst size of approximately 66 particles per infected bacterium. ΦImVa-1 remained stable for 2 h at a wide temperature (27-75 °C) and within a pH range of 5 to 10. Transmission electron microscopy revealed that ΦImVa-1 has an icosahedral head of approximately 60 nm in diameter with a short tail, resembling those in the Schitoviridae family. High throughput sequencing and bioinformatics analysis elucidated that ΦImVa-1 has a linear dsDNA genome of 77,479 base pairs (bp), with a G + C content of ~ 38.72% and 110 predicted gene coding regions (106 open reading frames and four tRNAs). The genome contains an extremely large virion-associated RNA polymerase gene and two smaller non-virion-associated RNA polymerase genes, which are hallmarks of schitoviruses. No antibiotic genes were found in the ΦImVa-1 genome. This is the first paper describing the biological properties, morphology, and the complete genome of a V. alginolyticus-infecting schitovirus. When raw mackerel fish flesh slices were treated with ΦImVa-1, the pathogen loads reduced significantly, demonstrating the potential of the phage as a biocontrol agent for V. alginolyticus strain ATCC 17749 in the food. KEY POINTS: • A novel schitovirus infecting Vibrio alginolyticus ATCC 17749 was isolated from Indian mackerel. • The complete genome of the phage was determined, analyzed, and compared with other phages. • The phage is heat stable making it a potential biocontrol agent in extreme environments.

Vibrio vulnificus, an Underestimated Zoonotic Pathogen.

V. vulnificus, continues being an underestimated yet lethal zoonotic pathogen. In this chapter, we provide a comprehensive review of numerous aspects of the biology, epidemiology, and virulence mechanisms of this poorly understood pathogen. We will emphasize the widespread role of horizontal gene transfer in V. vulnificus specifically virulence plasmids and draw parallels from aquaculture farms to human health. By placing current findings in the context of climate change, we will also contend that fish farms act as evolutionary drivers that accelerate species evolution and the emergence of new virulent groups. Overall, we suggest that on-farm control measures should be adopted both to protect animals from Vibriosis, and also as a public health measure to prevent the emergence of new zoonotic groups.

Comprehensive analysis of Vibrio alginolyticus: Environmental risk factors in the cultured Gilthead seabream (Sparus aurata) under seasonal fluctuations and water parameter alterations.

This study aimed to investigate the relationship between seasonal variations, water parameters and the prevalence of Vibriosis in Gilthead seabream. A total of 160 Gilthead seabream fish were sampled over the course of 1 year from private earthen pond farms in the Suez Canal area and examined for abnormalities and internal lesions. Vibrio alginolyticus, the causative agent of Vibriosis, was isolated and characterized from the sampled Gilthead seabream fish. The study revealed a significant correlation between different seasons and the prevalence of V. alginolyticus, with lower occurrence during autumn. Analysis of water parameters showed that toxic ammonia concentration was not effective in distinguishing between positive and negative cases of V. alginolyticus. Dissolved oxygen showed weak predictive ability for the occurrence of V. alginolyticus, while temperature demonstrated moderate potential as a predictor of its prevalence. pH values, organic matter concentrations and salinity showed no significant association with the occurrence of V. alginolyticus. Experimental challenges highlighted the vulnerability of Gilthead seabream to V. alginolyticus and emphasized the impact of environmental factors, such as pH and toxic ammonia, on their mortality and survival. The study emphasizes the importance of considering seasonal changes and water quality parameters in managing V. alginolyticus in mariculture. It underscores the need for careful monitoring and control of environmental factors to ensure the health and well-being of cultured fish populations. The findings contribute to our understanding of Vibriosis management and provide valuable insights for developing effective strategies in the aquaculture industry.

Cold temperature stress and damaged skin induced high mortality in barramundi (Lates calcarifer) challenged with Vibrio harveyi.

Most diseases in aquaculture are caused by opportunistic pathogens. One of them, Vibrio harveyi, is a widespread Gram-negative bacterium that has become an important pathogen of aquatic species in marine environments. Here, we propose the use of the causal pie model as a framework to conceptualize the causation of vibriosis in juvenile barramundi (Lates calcarifer) and to establish an effective challenge model. In the model, a sufficient cause, or the causal pie, is a constellation of component causes that lead to an outcome (e.g. vibriosis). In the pilot study, a high cumulative mortality (63.3% ± 10.0%, mean ± SE) was observed when V. harveyi was administered by intraperitoneal injection using a high challenge dose [107 colony-forming units (CFU) fish-1 ], but low or no mortality was observed in fish subject to cold stress or fish with intact skin when challenged by immersion. We, therefore, tested the use of a skin lesion (induced with a 4-mm biopsy punch) combined with cold temperature stress to induce vibriosis following the causal pie model. After challenge, fish were immediately subject to cold stress (22°C) or placed at an optimal temperature of 30°C. All groups were challenged with 108 CFU mL-1 for 60 min. A considerably higher mortality level (72.7% ± 13.9%) was observed in fish challenged with both a skin lesion and cold stress compared with mortality in fish only having a skin lesion (14.6% ± 2.8%). V. harveyi was re-isolated from all moribund fish and was detected by species-specific real-time PCR in gills, head kidney and liver, regardless of challenge treatment confirming vibriosis as the cause of disease. Parenchymal tissues had histopathological changes consistent with vibriosis. Whole-genome sequence (WGS) is provided for the Vibrio harveyi isolate examined in this study. Overall, the causal pie model was a useful framework to conceptualize the design of the experimental challenge model, in which both cold stress and skin damage were identified as component causes of vibriosis with high mortality. This conceptual framework can be applied to other opportunistic pathogens in aquaculture or to the study of co-infections in fish.

Management and Mitigation of Vibriosis in Aquaculture: Nanoparticles as Promising Alternatives.

Vibriosis is one of the most common diseases in marine aquaculture, caused by bacteria belonging to the genus Vibrio, that has been affecting many species of economically significant aquatic organisms around the world. The prevention of vibriosis in aquaculture is difficult, and the various treatments for vibriosis have their limitations. Therefore, there is an imperative need to find new alternatives. This review is based on the studies on vibriosis, specifically on the various treatments and their limitations, as well as the application of nanoparticles in aquaculture. One of the promising nanoparticles is graphene oxide (GO), which has been used in various applications, particularly in biological applications such as biosensors, drug delivery, and potential treatment for infectious diseases. GO has been shown to have anti-bacterial properties against both Gram-positive and Gram-negative bacteria, but no research has been published that emphasizes its impact on Vibrio spp. The review aims to explore the potential use of GO for treatment against vibriosis.

Effect of dietary celery (Apium graveolens) on the growth performance, immune responses, and bacterial resistance against Vibrio anguillarum of European seabass (Dicentrarchus labrax).

In this study, we evaluated to reveal the effects of aqueous methanolic extract of celery (Apium graveolens) on the growth performance, immune responses, and resistance against Vibrio anguillarum in European seabass (Dicentrarchus labrax). For this purpose, twenty fish (initial mean weight of 4.80 ± 0.06 g) were placed into twelve tanks (400 L) in triplicate and fish were fed with control (C) and three different levels (0.01, 0.05, and 0.1 g/kg) of A. graveolens (AG) extract-containing diets (AG0.01, AG0.05, and AG0.1) for 30 days. Blood and tissue (kidney, spleen, and intestine) samples were taken from the fish every 10 days during the study to determine the immune responses of the fish. Respiratory burst activity (RBA) was significantly decreased in the AG0.1 group compared to all other groups on the 10th day of the study (P < 0.05). Significance was noticed in the RBA of fish in all AG groups compared to the C group (P < 0.05) on the 30th day of the experiment Lysozyme activity (LYS) was raised on the 10th day of the study in all celery groups compared to the C group (P < 0.05). No differences in the myeloperoxidase activity (MPO) were observed among the experimental groups (P > 0.05). The final mean weight (FMW) was not affected in any experimental groups (P > 0.05). However, in the AG0.05 group, the specific growth rate (SGR) increased, and the feed conversion ratio (FCR) decreased compared to other groups (P < 0.05). IL-1ß in the kidney was highly elevated in the AG0.01 group on the 20th day of the study (P < 0.05). Similar results were observed on IL-6, IL-8, and TNF-α expression in the kidney (P < 0.05). Anti-inflammatory responses (IL-10 and TGF-ß) also increased in all experimental groups and tissues compared to the C group (P < 0.05). COX-2 was upregulated on the 20th day of the study in all tissues (P < 0.05). At the end of the feeding trial, the survival rate of the AG0.1 group in fish infected with Vibrio anguillarum infection was higher than the C group. Dietary celery extract did not affect growth performance directly but increased innate immune responses and a high survival rate. Overall, compared to the control group, the growth, immunity, and resistance of European seabass fed with a diet containing 0.05 g/kg celery aqueous methanolic extract has been improved, and this could be used as an immunostimulant feed additive.

Chitinolytic enzymes contribute to the pathogenicity of Aliivibrio salmonicida LFI1238 in the invasive phase of cold-water vibriosis.

BACKGROUND: Aliivibrio salmonicida is the causative agent of cold-water vibriosis in salmonids (Oncorhynchus mykiss and Salmo salar L.) and gadidae (Gadus morhua L.). Virulence-associated factors that are essential for the full spectrum of A. salmonicida pathogenicity are largely unknown. Chitin-active lytic polysaccharide monooxygenases (LPMOs) have been indicated to play roles in both chitin degradation and virulence in a variety of pathogenic bacteria but are largely unexplored in this context. RESULTS: In the present study we investigated the role of LPMOs in the pathogenicity of A. salmonicida LFI238 in Atlantic salmon (Salmo salar L.). In vivo challenge experiments using isogenic deletion mutants of the two LPMOs encoding genes AsLPMO10A and AsLPMO10B, showed that both LPMOs, and in particular AsLPMO10B, were important in the invasive phase of cold-water vibriosis. Crystallographic analysis of the AsLPMO10B AA10 LPMO domain (to 1.4 Å resolution) revealed high structural similarity to viral fusolin, an LPMO known to enhance the virulence of insecticidal agents. Finally, exposure to Atlantic salmon serum resulted in substantial proteome re-organization of the A. salmonicida LPMO deletion variants compared to the wild type strain, indicating the struggle of the bacterium to adapt to the host immune components in the absence of the LPMOs. CONCLUSION: The present study consolidates the role of LPMOs in virulence and demonstrates that such enzymes may have more than one function.

Non-serogroup O1/O139 agglutinable Vibrio cholerae: a phylogenetically and genealogically neglected yet emerging potential pathogen of clinical relevance.

Somatic antigen agglutinable type-1/139 Vibrio cholerae (SAAT-1/139-Vc) members or O1/O139 V. cholerae have been described by various investigators as pathogenic due to their increasing virulence potential and production of choleragen. Reported cholera outbreak cases around the world have been associated with these choleragenic V. cholerae with high case fatality affecting various human and animals. These virulent Vibrio members have shown genealogical and phylogenetic relationship with the avirulent somatic antigen non-agglutinable strains of 1/139 V. cholerae (SANAS-1/139- Vc) or O1/O139 non-agglutinating V. cholerae (O1/O139-NAG-Vc). Reports on implication of O1/O139-NAGVc members in most sporadic cholera/cholera-like cases of diarrhea, production of cholera toxin and transmission via consumption and/or contact with contaminated water/seafood are currently on the rise. Some reported sporadic cases of cholera outbreaks and observed change in nature has also been tracable to these non-agglutinable Vibrio members (O1/O139-NAGVc) yet there is a sustained paucity of research interest on the non-agglutinable V. cholerae members. The emergence of fulminating extraintestinal and systemic vibriosis is another aspect of SANAS-1/139- Vc implication which has received low attention in terms of research driven interest. This review addresses the need to appraise and continually expand research based studies on the somatic antigen non-serogroup agglutinable type-1/139 V. cholerae members which are currently prevalent in studies of water bodies, fruits/vegetables, foods and terrestrial environment. Our opinion is amassed from interest in integrated surveillance studies, management/control of cholera outbreaks as well as diarrhea and other disease-related cases both in the rural, suburban and urban metropolis.