Vibriosis In Fish Research Articles

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.

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.

Protective Effect of Willow (Salix babylonica L.) on Fish Resistance to Vibrio parahaemolyticus and Vibrio alginolyticus

Vibrio spp. cause vibriosis in many saltwater and freshwater aquatic species, such as fish, crustaceans, and mollusks. Vibrio parahaemolyticus and Vibrio alginolyticus are among the few Vibrio species commonly found in infections in fish. This study aimed at investigating the chemical composition and evaluating the antibacterial activities of Salix babylonica L. The ethyl acetate (LL2) and methanolic (LL3) extracts were used to evaluate the resistance of strains as V. parahaemolyticus LBT6 and VTCC 12233, and two strains of V. alginolyticus, NG20 and ATCC 17749, and compared their efficacy with cefotaxime in order to find an alternative to antibiotics in the treatment of vibriosis. The obtained results show that the LL2 extract, with its major components identified as chrysoeriol, luteolin, and β-sitosterol, exhibited a bacteriostatic effect against all the tested strains. In parallel, the LL3 extract, with the four major compounds luteolin-7-O-β-D-glucopyranoside, salicin, p-hydroxy benzoic acid, and β-sitosterol-3-O-β-D-glucopyranoside, showed significant bactericidal activity against these four strains; the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) varied from 2.0 to 3.0 μg/mL and from 3.5 to 5.0 μg/mL, respectively. Moreover, the LL3 extract could effectively increase the survival rate of the challenged fish at a dose of 5% (w/w) for the zebrafish (Danio rerio) and 3% (w/w) for the sea bass (Lates calcarifer). The LL3 extract showed a potential application of S. babylonica L. in the prevention and treatment of vibriosis in fish.

Vibrio-binding gangliosides in fish intestinal tracts.

It has been clarified that pathogens bind to glycosphingolipid (GSL) receptors in mammals, but there have been very few reports on pathogen-binding GSLs in fish. Vibrios are facultative anaerobic bacteria ubiquitous in marine and brackish environments. They are members of the normal intestinal microflora of healthy fish, but some species can cause a disease called vibriosis in fish and shellfish when the hosts are physiologically or immunologically weakened. The adherence of vibrios to host intestinal tracts is a significant event not only for survival and growth but also in terms of pathogenicity. We show in this mini-review that sialic acid-containing GSLs (gangliosides), GM4 and GM3, are receptors to which vibrios adhere to epithelial cells in the intestinal tract of fish. We also describe the enzymes responsible for synthesizing these Vibrio-binding gangliosides in fish.

Incidence of antibiotic resistance genotypes of Vibrio species recovered from selected freshwaters in Southwest Nigeria

Vibrio species are classified as potent hazards because of their tendency to effect serious diseases like cholera and other gastrointestinal ailments in humans, as well as vibriosis in fish. A total of 144 freshwater samples were aseptically collected monthly across four rivers (Asejire, Ona, Dandaru and Erinle rivers) over a 12-month period from which Vibrio spp. were isolated using culture procedures, confirmed by means of biochemical test as well as Polymerase Chain Reaction (PCR) assay and further characterized for their phenotypic antibiotic susceptibilities and relevant antimicrobial resistant determinants by PCR. Three hundred and fifteen (58%) isolates confirmed across the sampled sites (Asejire = 75, Dandaru = 87, Eleyele = 72, Erinle = 81) showed high resistance against erythromycin—95%, Sulphamethoxazole—94%, rifampicin—92%, doxycycline—82%, tetracycline—75%, amoxicillin—45%, cephalothin—43% and varied susceptibilities to other antibiotics. The multiple antibiotic resistance indices of 97% of the Vibrio isolates were above the 0.2 threshold limit with MAR phenotype pattern E-SUL-RF-TET-DOX (0.38) found to be the most prevalent pattern among the isolates. The distributions of resistance determinant of the tested antibiotics were revealed as follows: sulII 33%, sulI 19% (sulfonamides); blaOXA 27%, ampC 39%, blapse 11% (beta-lactams); tetA 28%, tetE 20%, tet39 8%, (tetracyclines) and strA 39%. aacC2 24%, aphA1 14% (aminoglycosides). Strong positive associations were observed among tetA, sulI, tetE and sulII. This study raises concerns as these selected rivers may contribute to the environmental spread of waterborne diseases and antibiotic resistance genes. Therefore, we recommend environmental context-tailored strategies for monitoring and surveillance of resistance genes so as to safeguard the environment from becoming reservoirs of virulent and infectious Vibrio species.

Genomics and transcriptomics reveal new molecular mechanism of vibriosis resistance in fish.

Infectious diseases have caused dramatic production decline and economic loss for fish aquaculture. However, the poor understanding of fish disease resistance severely hampered disease prevention. Chinese tongue sole (Cynoglossus semilaevis) is an important economic flatfish suffering from vibriosis. Here we used genomic, transcriptomic and experimental approaches to investigate the molecular genetic mechanisms underlying fish vibriosis resistance. A genome-wide comparison revealed that the genes under selective sweeps were enriched for glycosaminoglycan (GAG) chondroitin sulfate (CS)/dermatan sulfate (DS) metabolism. Transcriptomic analyses prioritized synergic gene expression patterns in this pathway, which may lead to an increased CS/DS content in the resistant family. Further experimental evidence showed that carbohydrate sulfotransferases 12 (Chst12), a key enzyme for CS/DS biosynthesis, has a direct antibacterial activity. To the best of our knowledge, this is the first report that the chst12 gene has a bactericidal effect. In addition, CS/DS is a major component of the extracellular matrix (ECM) and the selection signatures and fine-tuned gene expressions of ECM-receptor interaction genes indicated a modification in the ECM structure with an enhancement of the barrier function. Furthermore, functional studies conducted on Col6a2, encoding a collagen gene which constitutes the ECM, pointed to that it may act as a cellular receptor for Vibrio pathogens, thus plays an important role for the Vibrio invasion. Taken together, these findings provide new insights into the molecular protective mechanism underlying vibriosis resistance in fish, which offers crucial genomic resources for the resistant germplasm breeding and infectious disease control in fish culturing.

Corrigendum: A Transcriptomic Study Reveals That Fish Vibriosis Due to the Zoonotic Pathogen Vibrio vulnificus Is an Acute Inflammatory Disease in Which Erythrocytes May Play an Important Role

[This corrects the article DOI: 10.3389/fmicb.2022.852677.].

A Transcriptomic Study Reveals That Fish Vibriosis Due to the Zoonotic Pathogen Vibrio vulnificus Is an Acute Inflammatory Disease in Which Erythrocytes May Play an Important Role

Vibrio vulnificus is a marine zoonotic pathogen associated with fish farms that is considered a biomarker of climate change. Zoonotic strains trigger a rapid death of their susceptible hosts (fish or humans) by septicemia that has been linked to a cytokine storm in mice. Therefore, we hypothesize that V. vulnificus also causes fish death by triggering a cytokine storm in which red blood cells (RBCs), as nucleated cells in fish, could play an active role. To do it, we used the eel immersion infection model and then analyzed the transcriptome in RBCs, white BCs, and whole blood using an eel-specific microarray platform. Our results demonstrate that V. vulnificus triggers an acute but atypical inflammatory response that occurs in two main phases. The early phase (3 h post-infection [hpi]) is characterized by the upregulation of several genes for proinflammatory cytokines related to the mucosal immune response (il17a/f1 and il20) along with genes for antiviral cytokines (il12β) and antiviral factors (ifna and ifnc). In contrast, the late phase (12 hpi) is based on the upregulation of genes for typical inflammatory cytokines (il1β), endothelial destruction (mmp9 and hyal2), and, interestingly, genes related to an RNA-based immune response (sidt1). Functional assays revealed significant proteolytic and hemolytic activity in serum at 12 hpi that would explain the hemorrhages characteristic of this septicemia in fish. As expected, we found evidence that RBCs are transcriptionally active and contribute to this atypical immune response, especially in the short term. Based on a selected set of marker genes, we propose here an in vivo RT-qPCR assay that allows detection of early sepsis caused by V. vulnificus. Finally, we develop a model of sepsis that could serve as a basis for understanding sepsis caused by V. vulnificus not only in fish but also in humans.

Vibriosis in fish

This datasheet on vibriosis in fish covers Identity, Overview, Associated Diseases, Pests or Pathogens, Distribution, Hosts/Species Affected, Diagnosis, Pathology, Epidemiology, Impacts, Prevention/Control, Further Information.

Essential Oils Improve the Survival of Gnotobiotic Brine Shrimp (Artemia franciscana) Challenged With Vibrio campbellii.

The halophilic aquatic bacterium Vibrio campbellii is an important aquatic pathogen, capable of causing vibriosis in shrimp and fish resulting in significant economic losses. In a previous work, essential oils (EOs) extracts from Melaleuca alternifolia, Litsea citrata, and Eucalyptus citriodora were found to inhibit the growth of V. campbellii in vitro. This study aimed to determine in vivo EOs’ potential protective effect towards gnotobiotic brine shrimp Artemia franciscana, challenged with V. campbellii. The study showed that brine shrimp larvae supplemented with EOs of M. alternifolia (0.0008%) and L. citrata (0.002%) displayed significantly increased survival against V. campbellii. The results indicated that supplementation of these EOs increased the expression of immune-related genes (either in the presence or absence of the pathogen), probably contributing to enhanced protection. Furthermore, in vitro studies indicated that some EOs modulated the expression of virulence factors including swimming motility, biofilm formation, and gelatinase and lipase activity, while flow cytometry data and regrowth assay indicated that these EOs do not exhibit antimicrobial activity as V. campbellii grew at the tested concentrations [M. alternifolia (0.0008%) and L. citrata (0.002%)]. Our findings suggest that EOs extracted from M. alternifolia and L. citrata, can modulate virulence factor production and immunological responses and might hence become part of an intervention strategy to control vibriosis in a fish or shrimp aquaculture setting, a hypothesis that needs to be validated in the future.

Expression of LamB Vaccine Antigen in Wolffia globosa (Duck Weed) Against Fish Vibriosis.

Vibriosis is a commonly found bacterial disease identified among fish and shellfish cultured in saline waters. A multitude of Vibrio species have been identified as the causative agents. LamB, a member of outer membrane protein (OMPs) family of these bacteria is conserved among all Vibrio species and has been identified as an efficient vaccine candidate against vibriosis. Rootless duckweed (Wolffia) is a tiny, edible aquatic plant possessing characteristics suitable for the utilization as a bioreactor. Thus, we attempted to express a protective edible vaccine antigen against fish vibriosis in nuclear-transformed Wolffia. We amplified LamB gene from virulent Vibrio alginolyticus and it was modified to maximize the protein expression level and translocate the protein to the endoplasmic reticulum (ER) in plants. It was cloned into binary vector pMYC under the control of CaMV 35S promoter and introduced into Wolffia globosa by Agrobacterium-mediated transformation. Integration and expression of the LamB gene was confirmed by genomic PCR and RT-PCR. Western blot analysis revealed accumulation of the LamB protein in 8 transgenic lines. The cross-protective property of transgenic Wolffia was evaluated by orally vaccinating zebrafish through feeding fresh transgenic Wolffia and subsequently challenging with virulent V. alginolyticus. High relative percent survival (RPS) of the vaccinated fish (63.3%) confirmed that fish immunized with transgenic Wolffia were well-protected from Vibrio infection. These findings suggest that Wolffia expressed LamB could serve as an edible plant-based candidate vaccine model for fish vibriosis and feasibility of utilizing Wolffia as bioreactor to produce edible vaccines.

First screening report of immune and protective effect of non-toxic Jatropha vernicosa stem bark against Vibrio parahaemolyticus in Longfin yellowtail Seriola rivoliana leukocytes

In México, the infusion of Jatropha vernicosa stem bark has been used in folk medicine for many clinical situations, but no reports were available about this particular species of Jatropha in fish of mammals. In this first screening report, the phytochemical, antioxidant profile and antimicrobial properties of aqueous J. vernicosa stem bark extract were explored against Vibrio parahaemolyticus, an opportunist fish pathogen. To evaluate the cytotoxicity and immunological effect for the possible application of aqueous J. vernicosa stem bark in aquaculture, this study assessed it by using Longfin yellowtail Seriola rivoliana leukocytes. The results showed that phytochemical composition of the J. vernicosa extract was rich in phenol, flavonoid, saponin, and coumarin compounds. The antioxidant capacity of hydroxyl radical and superoxide anion scavenging activities, iron-chelation activity and β-carotene bleaching coupled to linoleic acid showed that J. vernicosa extracts had a moderate antioxidant effect compared with synthetic antioxidants (BHT, BHA and EDTA). No adverse effects were observed on spleen leukocytes (viability > 98%). Interestingly, J. vernicosa stem bark extract has immunostimulant and antioxidant effects, increasing phagocytosis, respiratory burns activity, and nitric oxide production, as well as superoxide dismutase and catalase activities. Additionally, J. vernicosa extract increased pro-inflammatory cytokine IL-1β and suppressed anti-inflammatory IL-10 gene expression upon stimuli and V. parahaemolyticus challenge. Finally, the data confirms that J. vernicosa stem bark extract is non-cytotoxic, rich in bioactive compounds with antioxidant effects, capable of enhancing the immune system in leukocytes and with great potential to fight against opportunistic diseases, such as vibriosis in fish.

Integrated transcriptomic and proteomic analyses of grass carp intestines after vaccination with a double-targeted DNA vaccine of Vibrio mimicus

Intestinal mucosal immunity plays a vital role against Vibrio mimicus infection because it is an enteric pathogen causing serious vibriosis in fish. In the previous studies, we developed an oral double-targeted DNA vaccine of V. mimicus and demonstrated that the vaccine could elicit significantly higher intestinal mucosal immune response than did naked DNA vaccine. But, little is known underlying regulatory molecular mechanisms of the enhanced intestinal mucosal immunity. Here the transcriptome and proteome in the intestines of the grass carps immunized or not with the double-targeted DNA vaccine were investigated by using RNA-seq and iTRAQ-coupled LC-MS/MS. Compared with the control group, a total of 5339 differentially expressed genes (DEGs) and 1173 differentially expressed proteins (DEPs) were identified in the immunized fish intestines. Subsequently, the integrated analysis between transcriptome and proteome data revealed that 250 DEPs were matched with the corresponding DEGs (named associated DEPs/DEGs) at both transcriptome and proteome levels. Fifty of all the associated DEPs/DEGs were immune-related and mainly enriched in phagosome, antigen-processing and presentation, complement and coagulation cascades, NLRs and MAPK signaling pathways via Gene Ontology and KEGG pathway analyses, which suggested the coordination of the five activated pathways was essential to the enhanced intestinal mucosal immune response in the immunized fish. The protein-protein interaction analysis showed that 60 of the 63 immune-related DEPs to form an integrated network. Additionally, randomly selected DEGs and DEPs were respectively validated by quantitative real-time RT-PCR and multiple reaction monitoring (MRM) assay, indicating that the both RNA-Seq and iTRAQ results in the study were reliable. Overall, our comprehensive transcriptome and proteome data provide some key genes and their protein products for further research on the regulatory molecular mechanisms underlying the enhanced intestinal mucosal immunity.

Large Phenotypic and Genetic Diversity of Prophages Induced from the Fish Pathogen Vibrio anguillarum.

Vibrio anguillarum is a marine pathogenic bacterium that causes vibriosis in fish and shellfish. Although prophage-like sequences have been predicted in V. anguillarum strains, many are not characterized, and it is not known if they retain the functional capacity to form infectious particles that can infect and lysogenize other bacterial hosts. In this study, the genome sequences of 28 V. anguillarum strains revealed 55 different prophage-related elements. Chemical and spontaneous induction allowed a collection of 42 phage isolates, which were classified in seven different groups according to a multiplex PCR assay. One shared prophage sequence, p41 (group III), was present in 17 V. anguillarum strains, suggesting that this specific element is very dynamically exchanged among V. anguillarum populations. Interestingly, the host range of genetically identical phages was highly dependent on the strains used for proliferation, indicating that phenotypic properties of phages were partly regulated by the host. Finally, experimental evidence displayed that the induced phage ɸVa_90-11-287_p41 was able to lysogenize V. anguillarum strain Ba35, and subsequently spontaneously become released from the lysogenized cells, demonstrating an efficient transfer of the phage among V. anguillarum strains. Altogether, the results showed large genetic and functional diversity and broad distribution of prophages in V. anguillarum, and demonstrated the potential of prophages as drivers of evolution in V. anguillarum strains.

The Expression of Virulence Factors in Vibrio anguillarum Is Dually Regulated by Iron Levels and Temperature.

Vibrio anguillarum causes a hemorrhagic septicemia that affects cold- and warm-water adapted fish species. The main goal of this work was to determine the temperature-dependent changes in the virulence factors that could explain the virulence properties of V. anguillarum for fish cultivated at different temperatures. We have found that although the optimal growth temperature is around 25°C, the degree of virulence of V. anguillarum RV22 is higher at 15°C. To explain this result, an RNA-Seq analysis was performed to compare the whole transcriptome profile of V. anguillarum RV22 cultured under low-iron availability at either 25 or 15°C, which would mimic the conditions that V. anguillarum finds during colonization of fish cultivated at warm- or cold-water temperatures. The comparative analysis of transcriptomes at high- and low-iron conditions showed profound metabolic adaptations to grow under low iron. These changes were characterized by a down-regulation of the energetic metabolism and the induction of virulence-related factors like biosynthesis of LPS, production of hemolysins and lysozyme, membrane transport, heme uptake, or production of siderophores. However, the expression pattern of virulence factors under iron limitation showed interesting differences at warm and cold temperatures. Chemotaxis, motility, as well as the T6SS1 genes are expressed at higher levels at 25°C than at 15°C. By contrast, hemolysin RTX pore-forming toxin, T6SS2, and the genes associated with exopolysaccharides synthesis were preferentially expressed at 15°C. Notably, at this temperature, the siderophore piscibactin system was strongly up-regulated. In contrast, at 25°C, piscibactin genes were down-regulated and the vanchrobactin siderophore system seems to supply all the necessary iron to the cell. The results showed that V. anguillarum adjusts the expression of virulence factors responding to two environmental signals, iron levels and temperature. Thus, the relative relevance of each virulence factor for each fish species could vary depending on the water temperature. The results give clues about the physiological adaptations that allow V. anguillarum to cause infections in different fishes and could be relevant for vaccine development against fish vibriosis.

Vibriosis in Fish: A Review on Disease Development and Prevention.

Current growth in aquaculture production is parallel with the increasing number of disease outbreaks, which negatively affect the production, profitability, and sustainability of the global aquaculture industry. Vibriosis is among the most common diseases leading to massive mortality of cultured shrimp, fish, and shellfish in Asia. High incidence of vibriosis can occur in hatchery and grow-out facilities, but juveniles are more susceptible to the disease. Various factors, particularly the source of fish, environmental factors (including water quality and farm management), and the virulence factors of Vibrio, influence the occurrence of the disease. Affected fish show weariness, with necrosis of skin and appendages, leading to body malformation, slow growth, internal organ liquefaction, blindness, muscle opacity, and mortality. A combination of control measures, particularly a disease-free source of fish, biosecurity of the farm, improved water quality, and other preventive measures (e.g., vaccination) might be able to control the infection. Although some control measures are expensive and less practical, vaccination is effective, relatively cheap, and easily implemented. In this review, the latest knowledge on the pathogenesis and control of vibriosis, including vaccination, is discussed.

Exploring the Effect of Phage Therapy in Preventing Vibrio anguillarum Infections in Cod and Turbot Larvae.

The aquaculture industry is suffering from losses associated with bacterial infections by opportunistic pathogens. Vibrio anguillarum is one of the most important pathogens, causing vibriosis in fish and shellfish cultures leading to high mortalities and economic losses. Bacterial resistance to antibiotics and inefficient vaccination at the larval stage of fish emphasizes the need for novel approaches, and phage therapy for controlling Vibrio pathogens has gained interest in the past few years. In this study, we examined the potential of the broad-host-range phage KVP40 to control four different V. anguillarum strains in Atlantic cod (Gadus morhua L.) and turbot (Scophthalmus maximus L.) larvae. We examined larval mortality and abundance of bacteria and phages. Phage KVP40 was able to reduce and/or delay the mortality of the cod and turbot larvae challenged with V. anguillarum. However, growth of other pathogenic bacteria naturally occurring on the fish eggs prior to our experiment caused mortality of the larvae in the unchallenged control groups. Interestingly, the broad-spectrum phage KVP40 was able to reduce mortality in these groups, compared to the nonchallenge control groups not treated with phage KVP40, demonstrating that the phage could also reduce mortality imposed by the background population of pathogens. Overall, phage-mediated reduction in mortality of cod and turbot larvae in experimental challenge assays with V. anguillarum pathogens suggested that application of broad-host-range phages can reduce Vibrio-induced mortality in turbot and cod larvae, emphasizing that phage therapy is a promising alternative to traditional treatment of vibriosis in marine aquaculture.

Phylogeny of Vibrio vulnificus from the Analysis of the Core-Genome: Implications for Intra-Species Taxonomy.

Vibrio vulnificus (Vv) is a multi-host pathogenic species currently subdivided into three biotypes (Bts). The three Bts are human-pathogens, but only Bt2 is also a fish-pathogen, an ability that is conferred by a transferable virulence-plasmid (pVvbt2). Here we present a phylogenomic analysis from the core genome of 80 Vv strains belonging to the three Bts recovered from a wide range of geographical and ecological sources. We have identified five well-supported phylogenetic groups or lineages (L). L1 comprises a mixture of clinical and environmental Bt1 strains, most of them involved in human clinical cases related to raw seafood ingestion. L2 is formed by a mixture of Bt1 and Bt2 strains from various sources, including diseased fish, and is related to the aquaculture industry. L3 is also linked to the aquaculture industry and includes Bt3 strains exclusively, mostly related to wound infections or secondary septicemia after farmed-fish handling. Lastly, L4 and L5 include a few strains of Bt1 associated with specific geographical areas. The phylogenetic trees for ChrI and II are not congruent to one another, which suggests that inter- and/or intra-chromosomal rearrangements have been produced along Vv evolution. Further, the phylogenetic trees for each chromosome and the virulence plasmid were also not congruent, which also suggests that pVvbt2 has been acquired independently by different clones, probably in fish farms. From all these clones, the one with zoonotic capabilities (Bt2-Serovar E) has successfully spread worldwide. Based on these results, we propose a new updated classification of the species based on phylogenetic lineages rather than on Bts, as well as the inclusion of all Bt2 strains in a pathovar with the particular ability to cause fish vibriosis, for which we suggest the name “piscis.”

Potential Outer Membrane Protein Candidates for Vaccine Development Against the Pathogen Vibrio anguillarum: A Reverse Vaccinology Based Identification.

Reverse vaccinology is a widely used approach that has facilitated the rapid identification of vaccine candidates suitable in vaccine development for pathogens. Vibrio anguillarum is a major pathogen responsible for vibriosis in fish and shellfish leading to huge economic losses to the aquaculture industry. Although commercial vaccines are available for fish against this bacterium they have their own limitations. In this study, we used the reverse vaccinology strategy to screen and identify V. anguillarum outer membrane proteins (OMPs) that could serve as vaccine candidates. Our analysis identified 23 antigenic outer membrane proteins which were highly conserved (>98% identity) across serovars of this bacterium. Of the 23, two were identified as outer membrane lipoproteins. Among the OMPs identified 18 were novel to this study and conserved across several Vibrio spp. with an identity of 21-93%. While the least (>48%) identity was observed for V. anguillarum ferrichrome-iron transporter protein, the highest identity (>80%) was seen for outer membrane proteins OmpK, BamA, OmpU, Fatty acid transporter, and two hypothetical proteins. These potential vaccine targets identified could contribute to the development of effective vaccine not only against V. anguillarum but also across other Vibrio spp. In addition, several B-cell and T-cell epitopes were predicted for the novel OMPs in this study which could aid in narrowing down peptide selection in designing a suitable epitope-based vaccine.

Isolation and Characterization of Large Marine Bacteriophage (Myoviridae), VhKM4 Infecting Vibrio harveyi.

The causative agent responsible for vibriosis in tropical fish aquaculture, Vibrio harveyi, has become a major bacterial pathogen. Studies suggest that this bacterium has developed resistance to antibiotics commonly used in aquaculture. In view of this situation and the requirement for the proposed postantibiotic era, bacteriophage therapy seems to be a promising control strategy for fish vibriosis. In this study, a lytic Vibrio phage VhKM4 belonging to a member of large, marine Myoviridae was successfully isolated. It exhibited bacteriolysis to both V. harveyi VHJR7 and V. parahaemolyticus ATCC 17802. The latent period of the VhKM4 phage was recorded at 60 min. It also recorded average burst size of approximately 52 plaque-forming units per infected cell. A strong bacteriolytic activity at low multiplicity of infection of 0.01 indicates the effectiveness of this large marine myovirid against fish pathogenic strain of V. harveyi VHJR7. Received June 16, 2016; accepted October 7, 2016.