Vibrio Tapetis Research Articles

D-amino acid peptides as antimicrobial agents against vibrio-associated diseases in aquaculture

Vibriosis is one of the most usual infection diseases in bivalve mollusks, particularly affecting seeds and larvae, which are more susceptible than adults to these bacterial infections. The devastating effect of vibriosis in shellfish hatcheries is aggravated by the increasing resistance of many Vibrio species to traditional antibiotics, highlighting the need to find new antimicrobial agents. Antimicrobial peptides (AMPs) are a promising alternative, however, their low stability can be a handicap for their practical application. In this study, the 3D structure and the stability of a synthetic all-D-amino acid peptide (D-Caerin) to proteases and extreme pH conditions have been analyzed and compared with its corresponding natural L-enantiomer peptide, Caerin 1.1. Moreover, the antimicrobial activity of D-Caerin has been tested in vitro against the control bacteria Micrococcus luteus CECT 245; and four Vibrio species: Vibrio aestuarianus CECT 625 T, Vibrio anguillarum CECT 522 T, Vibrio harveyi CECT 525 T and Vibrio tapetis CECT 4600 T, which are among the most representative causative agents of vibriosis in aquaculture. Our results demonstrate that D-Caerin contains two left-handed alpha helices, and is more stable and effective against the bacterial species tested than its corresponding natural L- counterpart. The bioactivity of D-Caerin has also been tested in vivo, in clam seeds infected with a mixed inoculum of these vibrio species. These preliminary assays show that D-Caerin-treated specimens exhibit higher survival rates after infection, demonstrating the superior stability of D-Caerin and its effectiveness against vibriosis-causative agents.

First Evidence of Vibrio tapetis as Causal Agent of Disease Outbreak in Meagre (Argyrosomus regius)

In this work, we studied a disease outbreak that affected meagre (Argyrosomus regius) at a temperature of 14–15 °C, and that reached a mortality rate of 45% in one week. Moribund fish showed neither external nor internal signs of disease. However, a large number of bacteria, phenotypically similar and in pure culture, were recovered from the liver and kidney. A further phenotypic, genotypic and serological characterization of a selection of three isolates (a828, a834 and a842) allowed the identification of them as Vibrio tapetis. Phylogenetic analysis placed the isolates within the subspecies tapetis, being clearly differentiated from the subspecies britannicus and quintayensis. Nevertheless, all the isolates showed some phenotypic discrepancies with the type strain CECT 4600T; also, they proved to belong to a different O-serotype and genogroup (according to REP-PCR analysis) than the type strain. A virulence evaluation performed in sea bass (Dicentrarchus labrax) by intraperitoneal injection using isolate a828 in two different doses (1 × 105 and 3 × 108 cfu/fish) led to mean mortality rates of 30% and 60%, respectively, corroborating its potential to cause significant mortalities in fish. To our knowledge, this is the first evidence of pathogenic Vibrio tapetis isolates associated with disease outbreaks in meagre.

Improving environmental monitoring of Vibrionaceae in coastal ecosystems through 16S rRNA gene amplicon sequencing

The Vibrionaceae family groups genetically and metabolically diverse bacteria thriving in all marine environments. Despite often representing a minor fraction of bacterial assemblages, members of this family can exploit a wide variety of nutritional sources, which makes them important players in biogeochemical dynamics. Furthermore, several Vibrionaceae species are well-known pathogens, posing a threat to human and animal health. Here, we applied the phylogenetic placement coupled with a consensus-based approach using 16S rRNA gene amplicon sequencing, aiming to reach a reliable and fine-level Vibrionaceae characterization and identify the dynamics of blooming, ecologically important, and potentially pathogenic species in different sites of the northern Adriatic Sea. Water samples were collected monthly at a Long-Term Ecological Research network site from 2018 to 2021, and in spring and summer of 2019 and 2020 at two sites affected by depurated sewage discharge. The 41 identified Vibrionaceae species represented generally below 1% of the sampled communities; blooms (up to ~ 11%) mainly formed by Vibrio chagasii and Vibrio owensii occurred in summer, linked to increasing temperature and particulate matter concentration. Pathogenic species such as Vibrio anguilllarum, Vibrio tapetis, and Photobacterium damselae were found in low abundance. Depuration plant samples were characterized by a lower abundance and diversity of Vibrionaceae species compared to seawater, highlighting that Vibrionaceae dynamics at sea are unlikely to be related to wastewater inputs. Our work represents a further step to improve the molecular approach based on short reads, toward a shared, updated, and curated phylogeny of the Vibrionaceae family.

Implication of the Type IV Secretion System in the Pathogenicity of Vibrio tapetis, the Etiological Agent of Brown Ring Disease Affecting the Manila Clam Ruditapes philippinarum.

Vibrio tapetis is a Gram-negative bacterium that causes infections of mollusk bivalves and fish. The Brown Ring Disease (BRD) is an infection caused by V. tapetis that primarily affects the Manila clam Ruditapes philippinarum. Recent studies have shown that a type IV secretion system (T4SS) gene cluster is exclusively found in strains of V. tapetis pathogenic to clams. However, whether the T4SS is implicated or not during the infection process remains unknown. The aim of this study was to create and characterize a V. tapetis T4SS null mutant, obtained by a near-complete deletion of the virB4 gene, in order to determine the role of T4SS in the development of BRD. This study demonstrated that the T4SS is neither responsible for the loss of hemocyte adhesion capacities, nor for the decrease of the lysosomal activity during BRD. Nevertheless, we observed a 50% decrease of the BRD prevalence and a decrease of mortality dynamics with the ΔvirB4 mutant. This work demonstrates that the T4SS of V. tapetis plays an important role in the development of BRD in the Manila clam.

MALDI-TOF MS as a promising tool to assess potential virulence of Vibrio tapetis isolates

Vibrio tapetis, the etiological agent of Brown Ring Disease, mainly affects the Manila clam Ruditapes philippinarum. Although this bacterium is mainly known as a clam pathogen, it has been isolated from several fish species. The main aim of the present study was to further explore the variability of 27 V. tapetis isolates from bivalves and fish, considering three different aspects; in vitro virulence based on the loss of clam hemocyte adhesion properties, detection of the gene virB4 encoding for an essential component of the Type IV Secretion System, and MALDI-TOF MS characterization based on whole cell extracts. Finally, these approaches were compared and evaluated for their ability to differentiate the potential pathogenicity of the 27 isolates against the Manila clams. Among the 11 V. tapetis isolates from the common dab isolated in 2018 in Belgium, only one (2BB) showed intermediate in vitro virulence against the Manila clam, and seven carried the virB4 gene while none of the V. tapetis previously isolated from fish in 2003 showed the presence of this particular gene. Finally, the peak protein profiles generated with MALDI-TOF MS analysis from all 27 V. tapetis strains showed a clear clustering of clam pathogenic and nonpathogenic isolates suggesting that a new isolate of V. tapetis that would cluster within the clam pathogenic isolates could be potentially pathogenic to the Manila clam. Thereupon, MALDI-TOF MS typing allows rapid and cost-efficient identification of V. tapetis isolates and can be defined as a complementary method of the traditional qPCR that opens new perspectives to study the virulence of V. tapetis isolates but also to perform environmental monitoring in order to prevent outbreaks.

External pH modulation during the growth of Vibrio tapetis, the aetiological agent of brown ring disease.

Brown ring disease (BRD) is an infection of the Manila clam Ruditapes philippinarum due to the pathogen Vibrio tapetis. During BRD, clams are facing immunodepression and shell biomineralization alteration. In this paper, we studied the role of pH on the growth of the pathogen and formulated hypothesis on the establishment of BRD by V. tapetis. In this study, we monitored the evolution of pH during the growth of V. tapetis in a range of pH and temperatures. We also measured the pH of Manila clam haemolymph and extrapallial fluids (EPFs) during infection by V. tapetis. We highlighted that V. tapetis modulates the external pH during its growth, to a value of 7·70. During the development of BRD, V. tapetis also influences EPFs and haemolymph pH in vitro in the first hours of exposure and in vivo after 3days of infection. Our experiments have shown a close interaction between V. tapetis CECT4600, a pathogen of Manila clam that induces BRD, and the pH of different compartments of the animals during infection. These results indicate that the bacterium, through a direct mechanism or as a consequence of physiological changes encountered in the animal during infection, is able to interfere with the pH of Manila clam fluids. This pH modification might promote the infection process or at least create an imbalance within the animal that would favour its persistence. This last hypothesis should be tested in future experiment. This study is the first observation of pH modifications in the context of BRD and might orient future research on the fine mechanisms of pH modulation associated with BRD.

A proteomic study of resistance to Brown Ring disease in the Manila clam, Ruditapes philippinarum

Marine mollusk aquaculture has more than doubled over the past twenty years, accounting for over 15% of total aquaculture production in 2016. Infectious disease is one of the main limiting factors to the development of mollusk aquaculture, and the difficulties inherent to combating pathogens through antibiotic therapies or disinfection have led to extensive research on host defense mechanisms and host-pathogen relationships. It has become increasingly clear that characterizing the functional profiles of response to a disease is an essential step in understanding resistance mechanisms and moving towards more effective disease control. The Manila clam, Ruditapes philippinarum, is a main cultured bivalve species of economic importance which is affected by Brown Ring disease (BRD), an infection induced by the bacterium Vibrio tapetis.In this study, juvenile Manila clams were subjected to a 28-day controlled challenge with Vibrio tapetis, and visual and molecular diagnoses were carried out to distinguish two extreme phenotypes within the experimental clams: uninfected (“RES”, resistant) and infected (“DIS”, diseased) post-challenge. Total protein extractions were carried out for resistant and diseased clams, and proteins were identified using LC-MS/MS. Protein sequences were matched against a reference transcriptome of the Manila clam, and protein intensities based on label-free quantification were compared to reveal 49 significantly accumulated proteins in resistant and diseased clams. Proteins with known roles in pathogen recognition, lysosome trafficking, and various aspects of the energy metabolism were more abundant in diseased clams, whereas those with roles in redox homeostasis and protein recycling were more abundant in resistant clams.Overall, the comparison of the proteomic profiles of resistant and diseased clams after a month-long controlled challenge to induce the onset of Brown Ring disease suggests that redox homeostasis and maintenance of protein structure by chaperone proteins may play important and interrelated roles in resistance to infection by Vibrio tapetis in the Manila clam.

Colony morphology and molecular identification of Vibrio spp. on green mussels (Perna viridis) in Yogyakarta, Indonesia tourism beach areas

Abstract. Hikmawati F, Susilowati A, Setyaningsih R. 2019. Colony morphology and molecular identification of Vibrio spp. on green mussels (Perna viridis) in Yogyakarta, Indonesia tourism beach areas. Biodiversitas 20: 2891-2899. Green mussels (Perna viridis) have filter feeder properties that allow pathogenic bacteria from the water environment to accumulate in relatively high levels. About 20% of foodborne diseases are caused by large quantities of seafood contaminated with bacteria. The purpose of this study is to determine the morphological characteristics, pathogenicity, identity, and the kinship of Vibrio species on green mussels in Yogyakarta coastal tourism areas. Vibrio spp. were grown on selective differential TCBS media. In this media, the suspected Vibrio spp. would produce yellow or green colonies. The ability of hemolysis of Vibrio was blood agar media, the species was molecularly identified using 16S rRNA gene sequence, and the phylogenetic relationship of the Vibrio spp., was analyzed using MEGA X Neighbor-Joining program. Based on morphological analysis, we obtained 23 bacterial isolates suspected to be Vibrio spp. Two Isolates (L1K2 6 and L2K2 13) were positive for α-hemolysis activity and 4 isolates (L1K1 3, L2K1 8, L2K2 16, and L3K2 22) were positive for β-hemolysis activity. The molecular analysis involved 18 Vibrio species, and 4 of them represented the Vibrio genus and 14 species represented 97-99% similarity species in accordance with the 16S rRNA sequence in database, namely: Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio cholerae, Vibrio neocaledonicus, Vibrio mimicus, Vibrio azureus, Vibrio diabolicus, Vibrio tapetis, Vibrio natriegens, and Vibrio owensii. The most dominant number of Vibrio isolates was V. alginolyticus, while the lowest was V. owensii. The highest number of Vibrio species in green mussels was found in Goa Cemara beach while the lowest was in Kwaru beach. Vibrio spp bacteria found in green clams in coastal tourism areas in Yogyakarta have close phylogenetic relationships with other Vibrio in seafood in Indonesian coastal waters.

Transcriptomic analysis of clam extrapallial fluids reveals immunity and cytoskeleton alterations in the first week of Brown Ring Disease development

The Brown Ring Disease is an infection caused by the bacterium Vibrio tapetis on the Manila clam Ruditapes philippinarum. The process of infection, in the extrapallial fluids (EPFs) of clams, involves alteration of immune functions, in particular on hemocytes which are the cells responsible of phagocytosis. Disorganization of the actin-cytoskeleton in infected clams is a part of what leads to this alteration. This study is the first transcriptomic approach based on collection of extrapallial fluids on living animals experimentally infected by V. tapetis. We performed differential gene expression analysis of EPFs in two experimental treatments (healthy-against infected-clams by V. tapetis), and showed the deregulation of 135 genes. In infected clams, a downregulation of transcripts implied in immune functions (lysosomal activity and complement- and lectin-dependent PRR pathways) was observed during infection. We also showed a deregulation of transcripts encoding proteins involved in the actin cytoskeleton organization such as an overexpression of β12-Thymosin (which is an actin sequestration protein) or a downregulation of proteins that closely interact with capping proteins such as Coactosin, that counteract action of capping proteins, or Profilin. We validated these transcriptomic results by cellular physiological analyses that showed a decrease of the lysosome amounts and the disorganization of actin cytoskeleton in infected hemocytes.

Molecular, transcriptional and functional delineation of Galectin-8 from black rockfish (Sebastes schlegelii) and its potential immunological role

Galectins are β-galactoside-binding lectins, which are involved in pattern recognition, cell adhesion, and stimulation of the host innate immune responses against microbial pathogens. In spite of several functional studies on different galectins isolated from vertebrates and invertebrates, this is the first report to present functional studies for galectin-8 from the marine teleost tissues. In the present study, we characterized galectin-8 homolog from black rockfish (Sebastes schlegelii), in molecular and functional aspects. Rockfish galectin-8 (SsGal8) was found to consist of a 969 bp long open reading frame (ORF), encoding a protein of 322 amino acids and the predicted molecular weight was 35.82 kDa. In silico analysis of SsGal8 revealed the presence of two carbohydrate binding domains (CRDs), at both N and C-termini and a linker peptide of 40 amino acids, in between the two domains. As expected, the phylogenetic tree categorized SsGal8 as a tandem-repeat galectin, and ultimately positioned it in the sub-clade of fish galectin-8. rSsGal8 was able to strongly agglutinate fish erythrocytes and the inhibition of agglutination was successfully exhibited by lactose and d-galactose. Bacterial agglutination assay resulted in agglutination of both Gram (+) and Gram (−) bacteria, including Escherichia coli, Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus parauberis, Lactococcus garvieae, Streptococcus iniae and Vibrio tapetis. The tissue distribution analysis based on qPCR assays, revealed a ubiquitous tissue expression of SsGal8 for the examined rockfish tissues, with the most pronounced expression in blood, followed by brain, intestine, head kidney and kidney. Furthermore, the mRNA transcription level of SsGal8 was significantly up-regulated in spleen, liver and head kidney, upon immune challenges with Streptococcus iniae, LPS and poly I:C, in a time dependent manner. Taken together, these findings strongly suggest the contribution of SsGal8 in regulating innate immune responses to protect the rockfish from bacterial infections.

Scrutinizing the triad of Vibrio tapetis, the skin barrier and pigmentation as determining factors in the development of skin ulcerations in wild common dab (Limanda limanda)

Recently, Vibrio tapetis was isolated for the first time from skin ulcerations in wild-caught common dab (Limanda limanda). To further examine its role in the development of these skin lesions, an in vivo experiment was performed. The significance of the skin barrier and in addition the difference between pigmented and non-pigmented side were investigated. Hence, the skin of common dab was treated in three different ways on both the pigmented and non-pigmented side. On a first “treatment zone”, the scales and overlying epidermal tissue were removed whereas in a second zone only the mucus was discarded. The third zone served as a non-treated zone. Thereafter, fish were challenged with V. tapetis. The control group was sham treated. Mortality, clinical signs, severity and size of the developing lesions were recorded. All animals were sacrificed and sampled 21 days post-inoculation. Significantly more fish of the group challenged with V. tapetis died compared to the control group with the highest incidence occurring 4 days post-inoculation. Fish challenged with V. tapetis developed more severe skin ulcerations. In zones where scales and epidermal tissue were removed, the ulcerations were more severe compared to zones where only mucus was eliminated. Ulcerations occurred more frequently, were more severe and larger on the pigmented side. Our data represents prove of V. tapetis as causative agent of ulcerative skin lesions although prior damage of the skin seems to be a major contributing factor. Furthermore, the pigmented side seemed predisposed to the development of skin ulcerations.

Relation between Biofilm and Virulence in Vibrio tapetis: A Transcriptomic Study.

Marine pathogenic bacteria are able to form biofilms on many surfaces, such as mollusc shells, and they can wait for the appropriate opportunity to induce their virulence. Vibrio tapetis can develop such biofilms on the inner surface of shells of the Ruditapes philippinarum clam, leading to the formation of a brown conchiolin deposit in the form of a ring, hence the name of the disease: Brown Ring Disease. The virulence of V. tapetis is presumed to be related to its capacity to form biofilms, but the link has never been clearly established at the physiological or genetic level. In the present study, we used RNA-seq analysis to identify biofilm- and virulence-related genes displaying altered expression in biofilms compared to the planktonic condition. A flow cell system was employed to grow biofilms to obtain both structural and transcriptomic views of the biofilms. We found that 3615 genes were differentially expressed, confirming that biofilm and planktonic lifestyles are very different. As expected, the differentially expressed genes included those involved in biofilm formation, such as motility- and polysaccharide synthesis-related genes. The data show that quorum sensing is probably mediated by the AI-2/LuxO system in V. tapetis biofilms. The expression of genes encoding the Type VI Secretion System and associated exported proteins are strongly induced, suggesting that V. tapetis activates this virulence factor when living in biofilm.

Vibrio tapetis Displays an Original Type IV Secretion System in Strains Pathogenic for Bivalve Molluscs.

The Brown Ring Disease (BRD) caused high mortality rates since 1986 in the Manila clam Venerupis philippinarum introduced and cultured in Western Europe from the 1970s. The causative agent of BRD is a Gram-Negative bacterium, Vibrio tapetis, which is also pathogenic to fish. Here we report the first assembly of the complete genome of V. tapetis CECT4600T, together with the genome sequences of 16 additional strains isolated across a broad host and geographic range. Our extensive genome dataset allowed us to describe the pathogen pan- and core genomes and to identify putative virulence factors. The V. tapetis core genome consists of 3,352 genes, including multiple potential virulence factors represented by haemolysins, transcriptional regulators, Type I restriction modification system, GGDEF domain proteins, several conjugative plasmids, and a Type IV secretion system. Future research on the coevolutionary arms race between V. tapetis virulence factors and host resistance mechanisms will improve our understanding of how pathogenicity develops in this emerging pathogen.

Autophagic processes in Mytilus galloprovincialis hemocytes: Effects of Vibrio tapetis

Autophagy is a highly conserved and regulated catabolic process involved in maintaining cell homeostasis in response to different stressors. The autophagic machinery is also used as an innate immune mechanism against microbial infection. In invertebrates, that lack acquired immunity, autophagy may thus play a key role in the protection against potential pathogens. In aquatic molluscs, evidence has been provided for induction of autophagy by starvation and different environmental stressors; however, no information is available on autophagic pathways in the immune cells, the hemocytes.In this work, the autophagic processes were investigated in the hemocytes of the marine bivalve, the mussel Mytilus galloprovincialis. The effects of classical inducers/inhibitors of mammalian autophagy were first tested. Rapamycin induced a decrease in lysosomal membrane stability-LMS that was prevented by the autophagy inhibitor Wortmannin. Increased MDC fluorescence and expression of LC3-II were also observed. Moreover, responses to in vitro challenge with the bivalve pathogen Vibrio tapetis were evaluated. Mussel hemocytes were unable to activate the immune response towards V. tapetis; however, bacterial challenge induced a moderate decrease in LMS, corresponding to lysosomal activation but no cytotoxicity; the effect was prevented by Wortmannin. TEM observations showed that V. tapetis resulted in rapid formation of autophagosomes and autolysosomes. Accordingly, increased LC3-II expression, decreased levels of phosphorylated mTor and of p62 were observed. The results represent the first evidence for autophagic processes in bivalve hemocytes in response to bacterial challenge, and underline the protective role of autophagy towards potential pathogenic vibrios.

First isolation of Vibrio tapetis and an atypical strain of Aeromonas salmonicida from skin ulcerations in common dab (Limanda limanda) in the North Sea.

Skin ulcerations rank amongst the most prevalent lesions affecting wild common dab (Limanda limanda) with an increase in prevalence of up to 3.5% in the Belgian part of the North Sea. A complex aetiology of these ulcerations is suspected, and many questions remain on the exact factors contributing to these lesions. To construct the aetiological spectrum of skin ulcerations in flatfish, a one-day monitoring campaign was undertaken in the North Sea. Fifteen fish presented with one or more ulcerations on the pigmented and/or non-pigmented side. Pathological features revealed various stages of ulcerations with loss of epidermal and dermal tissue, inflammatory infiltrates and degeneration of the myofibers bordering the ulceration, albeit in varying degrees. Upon bacteriological examination, pure cultures of Vibrio tapetis were retrieved in high numbers from five fish and of Aeromonas salmonicida in one fish. The V.tapetis isolates showed cross-reactivity with the sera against the representative strain of serotype O2 originating form a carpet-shell clam (Ruditapes descussatus). Moreover, the A.salmonicida isolates displayed a previously undescribed vapA gene sequence (A-layer type) with possible specificity towards common dab. Further research is necessary to pinpoint the exact role of these agents in the development of skin ulcerations in common dab.

Ancient DNA analysis identifies marine mollusc shells as new metagenomic archives of the past

Marine mollusc shells enclose a wealth of information on coastal organisms and their environment. Their life history traits as well as (palaeo-) environmental conditions, including temperature, food availability, salinity and pollution, can be traced through the analysis of their shell (micro-) structure and biogeochemical composition. Adding to this list, the DNA entrapped in shell carbonate biominerals potentially offers a novel and complementary proxy both for reconstructing palaeoenvironments and tracking mollusc evolutionary trajectories. Here, we assess this potential by applying DNA extraction, high-throughput shotgun DNA sequencing and metagenomic analyses to marine mollusc shells spanning the last ~7,000years. We report successful DNA extraction from shells, including a variety of ancient specimens, and find that DNA recovery is highly dependent on their biomineral structure, carbonate layer preservation and disease state. We demonstrate positive taxonomic identification of mollusc species using a combination of mitochondrial DNA genomes, barcodes, genome-scale data and metagenomic approaches. We also find shell biominerals to contain a diversity of microbial DNA from the marine environment. Finally, we reconstruct genomic sequences of organisms closely related to the Vibrio tapetis bacteria from Manila clam shells previously diagnosed with Brown Ring Disease. Our results reveal marine mollusc shells as novel genetic archives of the past, which opens new perspectives in ancient DNA research, with the potential to reconstruct the evolutionary history of molluscs, microbial communities and pathogens in the face of environmental changes. Other future applications include conservation of endangered mollusc species and aquaculture management.

Isolation of Vibrio tapetis from two native fish species (Genypterus chilensis and Paralichthys adspersus) reared in Chile and description of Vibrio tapetis subsp. quintayensis subsp. nov.

A group of seven Chilean isolates presumptively belonging to Vibrio tapetis was isolated from diseased fine flounders (Paralichthys adspersus) and red conger eel (Genypterus chilensis) experimentally reared in Quintay (Chile). All isolates were confirmed as members of V. tapetis on the basis of matrix-assisted laser desorption ionization time-of-flight MS, 16S rRNA gene sequencing, DNA-DNA hybridization values and G+C content. The ERIC-PCR and REP-PCR patterns were homogeneous among those isolates recovered from the same host (red conger or fine flounders), but distinct from the type strains V. tapetis subsp. tapetis CECT 4600T and V. tapetis subsp. britannicus CECT 8161T. On the basis of atpA, rpoA, rpoD, recA and pyrH gene sequence similarities (99.7-100 %) and clustering in the phylogenetic trees, the red conger isolates (Q20, Q047, Q48 and Q50) were confirmed as representing V. tapetis subsp. tapetis. However, they differed from V. tapetis subsp. tapetis CECT 4600T in their lipase, alpha quimiotripsin and non-acid phosphatase production. On the other hand, the fine flounder isolates (QL-9T, QL-35 and QL-41) showed rpoD, recA and pyrH gene sequence similarities ranging from 91.6 to 97.7 % with the type strains of the two V. tapetis subspecies (CECT 4600T and CECT 8161T) and consistently clustered together as an independent phylogenetic line within V. tapetis. Moreover, they could be differentiated phenotypically from strains CECT 4600T and CECT 8161T by nine and three different biochemical tests, respectively. In conclusion, the presence of V. tapetis in diseased red conger eel and fine flounder was demonstrated, extending the known host range and geographical location for this pathogen. Furthermore, this study demonstrates that the three isolates from fine flounder represent a novel subdivision within V. tapetis, for which the name V. tapetis subsp. quintayensis subsp. nov. is proposed and with QL-9T (=CECT 8851T=LMG 28759T) as the type strain. Although QL-9T was isolated from kidney of diseased fine flounder specimens, the challenge assays showed that it was non-pathogenic for this species.

Amoebic gill disease outbreak in marine fish cultured in Korea.

In 2015, 6.7-60% mortality was observed in black seabream ( Acanthopagrus schlegelii), rock bream ( Oplegnathus fasciatus), and gray mullet ( Mugil cephalus) farmed in the southern coast of Korea. On examination, numerous amoebae were found on the gills of these 3 fish species with detection rate of 100%. Some rock bream and gray mullet were coinfected with bacteria ( Pseudomonas anguilliseptica, Vibrio tapetis, or Vibrio anguillarum). Histologic examination revealed extensive hyperplastic epithelium and lamellar fusion in the gills. Numerous amoebae were seen between gill filaments. The amoebae collected from the 3 fish species had specific 630 bp of a partial 18S rRNA gene fragment for Neoparamoeba perurans. Phylogenetic analysis based on partial 18S rRNA gene nucleotide sequences revealed that these Korean amoeba isolates belonged to the N. perurans group. Based on our results, black seabream, rock bream, and gray mullet were added as new hosts for N. perurans.

Role of Vibrio tapetis in the development of skin ulceration in common dab (Limanda Limanda)

Role of Vibrio tapetis in the development of skin ulceration in common dab (Limanda Limanda)

Metabolic responses of clam Ruditapes philippinarum exposed to its pathogen Vibrio tapetis in relation to diet

We investigated the effect of brown ring disease (BRD) development and algal diet on energy reserves and activity of enzymes related to energy metabolism, antioxidant system and immunity in Manila clam, Ruditapes philippinarum. We found that algal diet did not impact the metabolic response of clams exposed to Vibrio tapetis. At two days post-injection (dpi), activities of superoxide dismutase and glutathione peroxidase (GPx) decreased whereas activities of nitric oxide synthase (iNOS) and catalase increased in infected clams, although no clinical signs were visible (BRD−). At 7 dpi, activities of several antioxidant and immune-related enzymes were markedly increased in BRD-likely indicating an efficient reactive oxygen species (ROS) scavenging compared to animals which developed clinical signs of BRD (BRD+). Therefore, resistance to BRD clinical signs appearance was associated with higher detoxification of ROS and enhancement of immune response. This study provides new biochemical indicators of disease resistance and a more comprehensive view of the global antioxidant response of clam to BRD development.