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Vibrio parahaemolyticus is a major seafood-borne zoonotic pathogen that causes gastroenteritis in humans and acute hepatopancreatic necrosis disease (AHPND) in shrimp. In this study, we isolated and characterized Vibrio phage vB_VpM-pA2SJ1, which infects clinical and AHPND-associated strains of V. parahaemolyticus. The phage genome is a linear dsDNA 51,054 bp in length with a G + C content of 43.7%, and it contains 89 open reading frames. Genome comparisons revealed basal similarity to other Vibrio phages, particularly Vibrio phage vB_VpP_1, with 84.2% identity and 46% coverage. Phylogenetic analysis based on the whole genome, the terminase large subunit, and the major capsid protein revealed that phage vB_VpM-pA2SJ1 did not cluster with other known phage families, thus indicating its uniqueness., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Background: The V. parahaemolyticus pandemic clone, results in the development of gastrointestinal illness in humans. Toxigenic strains of this species are frequently isolated from aquatic habitats and organisms such as mollusks and crustaceans. Reports on the isolation of the pandemic clone started in 1996, when a new O3:K6 clone was identified in Asia, that rapidly spread worldwide, becoming the predominant clone isolated from clinical cases. In this study whole genome sequencing was accomplished with an Illumina MiniSeq platform, upon six novel V. parahaemolyticus strains, that have been isolated in Mexico since 1998 and three representative genomes of strains that were isolated from reported outbreaks in other American countries, and were deposited in the GenBank. These nine genomes were compared against the reference sequence of the O3:K6 pandemic strain (RIMD 2210633), which was isolated in 1996, to determine sequence differences within American isolates and between years of isolation., Results: The results indicated that strains that were isolated at different times and from different countries, were highly genetically similar, among them as well as to the reference strain RIMD 2210633, indicating a high level of genetic stability among the strains from American countries between 1996 to 2012, without significant genetic changes relative to the reference strain RIMD 2210633, which was isolated in 1996 and was considered to be representative of a novel O3:K6 pandemic strain., Conclusions: The genomes of V. parahaemolyticus strains isolated from clinical and environmental sources in Mexico and other American countries, presented common characteristics that have been reported for RIMD 2210633 O3:K6 pandemic strain. The major variations that were registered in this study corresponded to genes non associated to virulence factors, which could be the result of adaptations to different environmental conditions. Nevertheless, results do not show a clear pattern with the year or locality where the strains were isolated, which is an indication of a genomic stability of the studied strains., (© 2021. The Author(s).)

Vibrio parahaemolyticus is a major foodborne pathogen and is also pathogenic to shrimp. Due to the emergence of multidrug-resistant V. parahaemolyticus strains, bacteriophages have shown promise as antimicrobial agents that could be used for controlling antibiotic-resistant strains. Here, a V. parahaemolyticus phage, vB_VpaP_MGD2, was isolated from a clam (Meretrix meretrix) and further characterized to evaluate its potential capability for biocontrol. Podophage vB_VpaP_MGD2 had a wide host range and was able to lyse 27 antibiotic-resistant V. parahaemolyticus strains. A one-step growth curve showed that vB_VpaP_MGD2 has a short latent period of 10 min and a large burst size of 244 phages per cell. Phage vB_VpaP_MGD2 was able to tolerate a wide range of temperature (30 °C-50 °C) and pH (pH 3-pH 10). Two multidrug-resistant strains (SH06 and SA411) were suppressed by treatment with phage vB_VpaP_MGD2 at a multiplicity of infection of 100 for 24 h without apparent regrowth of bacterial populations. The frequency of mutations causing bacteriophage resistance was relatively low (3.1 × 10 -6 ). Phage vB_VpaP_MGD2 has a double-stranded DNA with a genome size of 45,105 bp. Among the 48 open reading frames annotated in the genome, no lysogenic genes or virulence genes were detected. Sequence comparisons suggested that vB_VpaP_MGD2 is a member of a new species in the genus Zindervirus within the subfamily Autographivirinae. This is the first report of a member of the genus Zindervirus that can infect V. parahaemolyticus. These findings suggest that vB_VpaP_MGD2 may be a candidate biocontrol agent against early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) caused by multidrug-resistant V. parahaemolyticus in shrimp production.

This study investigated the effects of replacement of fishmeal (FM) with poultry by-product (PBM) protein, supplemented with black soldier fly, Hermetia illucens (HI) larvae on growth, histomormhology, immunity and resistance to Vibrio harveyi in juvenile barramundi. Two hundred and twenty five barramundi averaging 3.51 ± 0.03 g were randomly allocated into three groups and fed isonitrogenous and isocalorific diets containing different levels of PBM supplemented with HI as follows: Control (FM based diet), 45PBM + HI (45% PBM supplemented with 10% HI), and 90PBM + HI (90% PBM supplemented with 10% HI) for 6 weeks. Results showed that dietary inclusion of 45PBM + HI significantly improved the growth performance than control whereas growth inhibition occurred in the 90PBM + HI. The 45PBM + HI groups demonstrated significant increases in histometric measurements (villus and enterocyte width, and microvilli height) and acidic mucins. The impaired growth in 90PBM + HI groups was further associated with multifocal necrosis in the liver, an upregulation of the stress related genes (HSP70 and HSP90) and increase in the levels of liver enzymes. When 45PBM + HI was fed, survival against V. harveyi increased significantly and also an increase in serum immunity and immune-related genes in the head kidney was observed after infection.

Vibrio vulnificus is an opportunistic human pathogen responsible for the majority of seafood-associated deaths worldwide and is also a relevant fish pathogen for the aquaculture industry. In addition to infections in aquatic livestock, V. vulnificus also represents a risk to aquarium animals. For the first time, this work describes an important mortality outbreak in Trachinotus goodei in a zoo aquarium, with the isolation of Vibrio vulnificus (Vv) from the internal organs of the diseased fish. The isolates were identified by MALDI-TOF MS, serotyped and characterized by pulsed-field gel electrophoresis (PFGE). Although the isolates from great pompanos did not belong to pathovar piscis (formerly biotype 2) or to any of the fish-related serovars, they all had identical phenotypes, antimicrobial susceptibility profiles and PFGE patterns, which together with their isolation in pure culture from internal organs is strongly indicative of their clinical significance. Moreover, Vv isolates harboured important genetic markers of human virulence potential: they had the clinical variant of the vcg gene, gave the 338 bp DNA amplification product of the pilF gene and resisted the bactericidal activity of human serum. All these results strongly suggest that these Vv isolates should be considered potentially virulent for humans. These results extend the range of fish species affected by V. vulnificus, confirm the threat that this pathogen represents to aquatic animals and highlight the risk that this bacterial pathogen poses to human health., (© 2019 Blackwell Verlag GmbH.)

There is an increasing emergence of antibiotic-resistant Vibrio alginolyticus, a zoonotic pathogen that causes mass mortality in aquatic animals and infects humans; therefore, there is a demand for alternatives to antibiotics for the treatment and prevention of infections caused by this pathogen. One possibility is through the exploitation of bacteriophages. In the present study, the novel bacteriophage pVa-21 was classified as Myoviridae and characterised as a candidate biocontrol agent against V. alginolyticus. Its morphology, host range and infectivity, growth characteristics, planktonic or biofilm lytic activity, stability under various conditions, and genome were investigated. Its latent period and burst size were estimated to be approximately 70 min and 58 plaque-forming units/cell, respectively. In addition, phage pVa-21 can inhibit bacterial growth in both the planktonic and biofilm states. Furthermore, phylogenetic and genome analysis revealed that the phage is closely related to the giant phiKZ-like phages and can be classified as a new member of the phiKZ-like bacteriophages that infect bacteria belonging to the family Vibrionaceae.

As an opportunist pathogen, Vibrio alginolyticus (V. alginolyticus), causes disease in marine animals. Bacterial contamination of seafood is not uncommon, and phage therapy is considered a safe way to decontaminate such foods to control the emergence of vibriosis. Here, we report on the isolation of a new, virulent phage called vB&#95;ValP&#95;IME271 (designated phage IME271), which infects V. alginolyticus and was isolated from seawater. Phage IME271 displayed good pH (7-9) and temperature tolerance (< 40 °C) and had a broad host range against Vibrio isolates, including 7 strains of V. alginolyticus and11 strains of V. parahaemolyticus. The IME271 genome was sequenced and annotated, the results of which showed that this phage is a Podoviridae family member with a genome length of 50,345 base pairs. The complete genome is double-stranded DNA with a G+C content of 41.4%. Encoded within the genome are 67 putative proteins, of which only 22 coding sequences have known functions, and no tRNAs are present. The BLASTn results for IME271 showed that it only shares similarity with the Vibrio phage VPp1 (sequence identity score of 96% over 87% of the genome) whose host is V. parahaemolyticus. Comparative analysis showed that IME271 and VPp1 share a similar genomic structure, and the structural proteins are highly similar (> 95% similarity score). In summary, our work identified a new lytic Podoviridae bacteriophage, which is infective to V. alginolyticus and V. parahaemolyticus. This bacteriophage could potentially be used to control V. alginolyticus and V. parahaemolyticus infections in marine animals.

Bacterial diseases cause high mortality in Penaeus (Litopenaeus) vannamei postlarvae. Therefore, appropriate application of efficient therapeutic products is of vital importance for disease control. This study evaluated through in vitro analyses the antimicrobial effectiveness of commercial therapeutic products used for P. vannamei bacterial diseases and antibiotics against pathogenic Vibrio strains circulating in Ecuadorian hatcheries. Twenty strains were isolated from 31 larvae samples with high bacterial counts from 10 hatcheries collected during mortality events. The strains virulence was verified through challenge tests with Artemia franciscana nauplii and P. vannamei postlarvae. Through 16S rRNA sequence analysis, strains showed a great similarity to the Vibrio sequences reported as pathogens, with 95% belonging to the Harveyi clade. Through antibiograms and minimal inhibitory concentration (MIC) in vitro tests we found that furazolidone, ciprofloxacin, chloramphenicol, norfloxacin, nalidixic acid, florfenicol, fosfomycin and enrofloxacin inhibited the growth of all or most of the strains. Less efficient antibiotics were penicillin, oxytetracycline and tetracycline. A multiple antibiotic resistance (MAR) index of 0.23 showed some level of resistance to antibiotics, with two MAR prevalent patterns (Penicillin-Oxytetracycline and Penicillin-Oxytetracycline-Tetracycline). From a total of 16 natural products (five probiotics, nine organic acids and two essential oils), only three (one probiotic, one organic acid and one essential oil) were effective to control most of the strains. Shrimp producers can apply relatively simple in vitro analyses, such as those employed in this study, to help take adequate management decisions to reduce the impact of bacterial diseases and increase profit., Competing Interests: The authors have declared that no competing interests exist.

The Notch signaling pathway transcriptional regulator, CSL (also called as CBF1, Suppressor of Hairless or Lag-1 in different species, generally designated as CSL1), is not only associated with cell proliferation and differentiation but also involved in tumorigenesis, inflammation and immune regulation in vertebrates. We recently showed that Notch signaling was involved in the immune response of Litopenaeus vannamei shrimp. However, as an important transcriptional regulator of this pathway, whether or not shrimp CSL was also involved in immune response had not been explored. Here, we cloned and characterized the CSL gene in L. vannamei (LvCSL), which has a 2271 bp open reading frame (ORF) encoding a putative protein of 756 amino acids, and contains two conserved Lag1-DNA bind as well as beta trefoil domains (BTD). LvCSL clustered with invertebrates in the phylogenetic tree and closely related to the RBP Jk X1 of Parasteatoda tepidariorum. The transcript level of LvCSL analyzed by quantitative polymerase chain reaction (qPCR) showed that LvCSL was widely expressed in all tissues tested, with induced levels observed in the hepatopancreas and hemocytes following immune challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), therefore, suggesting LvCSL involvement in shrimp immune response to pathogens. Besides, LvCSL knockdown decreased the expression of proliferation-related genes (LvHey2 and LvAstakine), and attenuated the expression of immune-related genes L. vannamei hypoxia inducible factor alpha (LvHIF-α), LvLectin and L. vannamei small subunit hemocyanin (LvHMCS) in shrimp hemocytes, as well as significantly decreased total hemocyte count. Moreover, high cumulative mortality was observed in LvCSL depleted shrimp challenged with V. parahaemoliticus. In conclusion, our present data strongly suggest that LvCSL is an important factor in shrimp, vital for shrimp survival and contributing to immune resistance to pathogens., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The transforming growth factor-β (TGF-β) receptor-mediated TGF-β signaling cascade plays important roles in diverse cellular processes, including cell proliferation, differentiation, growth, apoptosis and inflammation in vertebrates. In the present study, the type I TGF-β receptor (TβR1) was firstly identified and characterized in mud crab Scylla paramamosain. The full-length cDNA of SpTβR1 was 1, 986 bp with a 1, 608 bp open reading frame, which encoded a putative protein of 535 amino acids including a typical transmembrane region, a conserved glycine-serine (GS) motif and a S&#95;TKc domain (Serine/Threonine protein kinases, catalytic domain). Real-time PCR analysis showed that SpTβR1 was predominantly expressed at early embryonic development stage and was highly expressed at postmolt stages during molt cycle, suggesting its participation in development and growth. Moreover, the expression levels of SpTβR1 in hepatopancreas and hemocytes were positively induced after the challenges of Vibro alginolyticus and Poly (I:C), indicating the involvement of SpTβR1 in responding to both bacterial and viral infections. The in vivo RNA interference assays demonstrated that the expression levels of two NF-κB members (SpRelish and SpDorsal) and six antimicrobial peptide (AMP) genes (SpCrustin and SpALF2-6) were significantly suppressed when the SpTβR1 was silenced. Additionally, the expression levels of SpTβR1, SpRelish, SpDorsal and AMPs were consistently down-regulated or up-regulated when the primary cultured hemocytes were treated with TβR1 antagonist or agonist for 24 h. These results indicated that TβR1 not only contributed to the crabs' development and growth but also played vital role in the innate immunity of S. paramamosain, and it also provided new insights into the origin or evolution of TGF-β receptors in crustacean species and even in invertebrates., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Acute hepatopancreatic necrosis disease (AHPND) has extended rapidly, causing alarming shrimp mortalities. Initially, the only known causative agent was Vibrio parahaemolyticus carrying a plasmid coding for the mortal toxins Pir VP . Recently, it has been found that the plasmid and hence the disease, could be transferred among members of the Harveyi clade. The current study performs a genomic characterization of an isolate capable of developing AHPND in shrimp. Mortality studies and molecular and histopathological analyses showed the infection capacity of the strain. Multilocus sequence analysis placed the bacteria as a member of the Orientalis clade, well known for containing commensal and even probiotic bacteria used in the shrimp industry. Further whole genome comparative analyses, including Vibrio species from the Orientalis clade, and phylogenomic metrics (TETRA, ANI and DDH) showed that the isolate belongs to a previously unidentified species, now named Vibrio punensis sp. nov. strain BA55. Our findings show that the gene transfer capacity of Vibrio species goes beyond the clade classification, demonstrating a new pathogenic capacity to a previously known commensal clade. The presence of these genes in a different Vibrio clade may contribute to the knowledge of the Vibrio pathogenesis and has major implications for the spread of emerging diseases.

Background: Evolutionary shifts in bacterial virulence are often associated with a third biological player, for instance temperate phages, that can act as hyperparasites. By integrating as prophages into the bacterial genome they can contribute accessory genes, which can enhance the fitness of their prokaryotic carrier (lysogenic conversion). Hyperparasitic influence in tripartite biotic interactions has so far been largely neglected in empirical host-parasite studies due to their inherent complexity. Here we experimentally address whether bacterial resistance to phages and bacterial harm to eukaryotic hosts is linked using a natural tri-partite system with bacteria of the genus Vibrio, temperate vibriophages and the pipefish Syngnathus typhle. We induced prophages from all bacterial isolates and constructed a three-fold replicated, fully reciprocal 75 × 75 phage-bacteria infection matrix., Results: According to their resistance to phages, bacteria could be grouped into three distinct categories: highly susceptible (HS-bacteria), intermediate susceptible (IS-bacteria), and resistant (R-bacteria). We experimentally challenged pipefish with three selected bacterial isolates from each of the three categories and determined the amount of viable Vibrio counts from infected pipefish and the expression of pipefish immune genes. While the amount of viable Vibrio counts did not differ between bacterial groups, we observed a significant difference in relative gene expression between pipefish infected with phage susceptible and phage resistant bacteria., Conclusion: These findings suggest that bacteria with a phage-susceptible phenotype are more harmful against a eukaryotic host, and support the importance of hyperparasitism and the need for an integrative view across more than two levels when studying host-parasite evolution.

The major histocompatibility complex (MHC) genes are crucial in the adaptive immune system, and the gene duplication of MHC in animals can generally result in immune flexibility. In this study, we found that the lined seahorse (Hippocampus erectus) has only one gene copy number (GCN) of MHC IIα and IIβ, which is different from that in other teleosts. Together with the lack of spleen and gut-associated lymphatic tissue (GALT), the seahorse may be referred to as having a partial but natural "immunodeficiency". Highly variable amino acid residues were found in the IIα and IIβ domains, especially in the α1 and β1 domains with 9.62% and 8.43% allelic variation, respectively. Site models revealed seven and ten positively selected positions in the α1 and β1 domains, respectively. Real-time PCR experiments showed high expression levels of the MHC II genes in intestine (In), gill (Gi) and trunk kidney (TK) and medium in muscle (Mu) and brood pouch (BP), and the expression levels were significantly up-regulated after bacterial infection. Specially, relative higher expression level of both MHC IIα and IIβ was found in Mu and BP when compared with other fish species, in which MHC II is expressed negligibly in Mu. These results indicate that apart from TK, Gi and In, MU and BP play an important role in the immune response against pathogens in the seahorse. In conclusion, high allelic variation and strong positive selection in PBR and relative higher expression in MU and BP are speculated to partly compensate for the immunodeficiency., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Vibrio parahaemolyticus is a Gram-negative, motile, nonspore-forming pathogen that causes foodborne illness associated with the consumption of contaminated seafoods. Although many cases of foodborne outbreaks caused by V. parahaemolyticus have been reported, the genomes of only five strains have been completely sequenced and analyzed using bioinformatics. In order to characterize overall virulence factors and pathogenesis of V. parahaemolyticus associated with foodborne outbreak in South Korea, a new strain FORC&#95;008 was isolated from flounder fish and its genome was completely sequenced. The genomic analysis revealed that the genome of FORC&#95;008 consists of two circular DNA chromosomes of 3266 132 bp (chromosome I) and 1772 036 bp (chromosome II) with a GC content of 45.36% and 45.53%, respectively. The entire genome contains 4494 predicted open reading frames, 129 tRNAs and 31 rRNA genes. While the strain FORC&#95;008 does not have genes encoding thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), its genome encodes many other virulence factors including hemolysins, pathogenesis-associated secretion systems and iron acquisition systems, suggesting that it may be a potential pathogen. This report provides an extended understanding on V. parahaemolyticus in genomic level and would be helpful for rapid detection, epidemiological investigation and prevention of foodborne outbreak in South Korea., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Vibrio parahaemolyticus is the most common cause of seafood-borne gastroenteritis worldwide and a blight on global aquaculture. This organism requires a horizontally acquired type III secretion system (T3SS2) to infect the small intestine, but knowledge of additional factors that underlie V. parahaemolyticus pathogenicity is limited. We used transposon-insertion sequencing to screen for genes that contribute to viability of V. parahaemolyticus in vitro and in the mammalian intestine. Our analysis enumerated and controlled for the host infection bottleneck, enabling robust assessment of genetic contributions to in vivo fitness. We identified genes that contribute to V. parahaemolyticus colonization of the intestine independent of known virulence mechanisms in addition to uncharacterized components of T3SS2. Our study revealed that toxR, an ancestral locus in Vibrio species, is required for V. parahaemolyticus fitness in vivo and for induction of T3SS2 gene expression. The regulatory mechanism by which V. parahaemolyticus ToxR activates expression of T3SS2 resembles Vibrio cholerae ToxR regulation of distinct virulence elements acquired via lateral gene transfer. Thus, disparate horizontally acquired virulence systems have been placed under the control of this ancestral transcription factor across independently evolved human pathogens.

Foodborne outbreaks caused by a mixed infection of Vibrio parahaemolyticus and norovirus have rarely been described. We reported a mixed outbreak of Vibrio parahaemolyticus and norovirus causing acute gastroenteritis in 99 staff members of a company in Guangdong, China, in May 2013, following consumption of roasted duck, an uncommon non-seafood vehicle for such mixed infection, in one meal served in the company's catering service. Epidemiological and laboratory findings indicated that a single asymptomatic food handler was the source of both pathogens, and the high rate of infection of both pathogens was exacerbated by the setting's suboptimal food hygiene practice.

An inflammatory response is a hallmark of necrosis evoked by bacterial pathogens. Vibrio vulnificus, VvpE, is an elastase that is responsible for tissue necrosis and inflammation; however, the molecular mechanism by which it regulates host cell death has not been characterized. In the present study, we investigate the cellular mechanism of VvpE with regard to host cell death and the inflammatory response of human intestinal epithelial (INT-407) cells. The recombinant protein (r)VvpE (50 pg/ml) caused cytotoxicity mainly via necrosis coupled with IL-1β production. The necrotic cell death induced by rVvpE is highly susceptible to the knockdown of annexin A (ANXA)2 and the sequestration of membrane cholesterol. We found that rVvpE induces the recruitment of NADPH oxidase 2 and neutrophil cytosolic factor 1 into membrane lipid rafts coupled with ANXA2 to facilitate the production of reactive oxygen species (ROS). The bacterial signaling of rVvpE through ROS production is uniquely mediated by the phosphorylation of redox-sensitive transcription factor NF-κB. The silencing of NF-κB inhibited IL-1β production during necrosis. rVvpE induced hypomethylation and region-specific transcriptional occupancy by NF-κB in the IL-1β promoter and has the ability to induce pyroptosis via NOD-, LRR-, and pyrin domain-containing 3 inflammasome. In a mouse model of V. vulnificus infection, the mutation of the vvpE gene from V. vulnificus negated the proinflammatory responses and maintained the physiological levels of the proliferation and migration of enterocytes. These results demonstrate that VvpE induces the hypomethylation of the IL-1β promoter and the transcriptional regulation of NF-κB through lipid raft-dependent ANXA2 recruitment and ROS signaling to promote IL-1β production in intestinal epithelial cells., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Bacterial strains used as backbone for the generation of vaccine prototypes should exhibit an adequate and stable safety profile. Given the fact that live attenuated vaccines often contain some potential risks in virulence recovery and spread infections, new approaches are greatly needed to improve their biological safety. Here, a critically iron-regulated promoter PviuA was screened from Vibrio anguillarum, which was demonstrated to respond to iron-limitation signal both in vitro and in vivo. By using PviuA as a regulatory switch to control the expression of phage P22 lysis cassette 13-19-15, a novel in vivo inducible bacterial lysis system was established in V. anguillarum. This system was proved to be activated by iron-limitation signals and then effectively lyse V. anguillarum both in vitro and in vivo. Further, this controllable bacterial lysis system, after being transformed into a live attenuated V. anguillarum vaccine strain MVAV6203, was confirmed to significantly improve biological safety of the live attenuated vaccine without impairing its immune protection efficacy., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Vibrio parahaemolyticus is the leading worldwide cause of seafood-associated gastroenteritis, yet little is known regarding its intraintestinal gene expression or physiology. To date, in vivo analyses have focused on identification and characterization of virulence factors--e.g. a crucial Type III secretion system (T3SS2)--rather than genome-wide analyses of in vivo biology. Here, we used RNA-Seq to profile V. parahaemolyticus gene expression in infected infant rabbits, which mimic human infection. Comparative transcriptomic analysis of V. parahaemolyticus isolated from rabbit intestines and from several laboratory conditions enabled identification of mRNAs and sRNAs induced during infection and of regulatory factors that likely control them. More than 12% of annotated V. parahaemolyticus genes are differentially expressed in the intestine, including the genes of T3SS2, which are likely induced by bile-mediated activation of the transcription factor VtrB. Our analyses also suggest that V. parahaemolyticus has access to glucose or other preferred carbon sources in vivo, but that iron is inconsistently available. The V. parahaemolyticus transcriptional response to in vivo growth is far more widespread than and largely distinct from that of V. cholerae, likely due to the distinct ways in which these diarrheal pathogens interact with and modulate the environment in the small intestine., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Foodborne disease caused by pathogenic Vibrio parahaemolyticus has become a serious public health problem in many countries. Rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. In this study, an EvaGreen-based multiplex real-time PCR assay was established for the detection of pathogenic V. parahaemolyticus. This assay targeted three genetic markers of V. parahaemolyticus (species-specific gene toxR and virulence genes tdh and trh). The assay could unambiguously identify pathogenic V. parahaemolyticus with a minimum detection limit of 1.4 pg genomic DNA per reaction (concentration giving a positive multiplex real-time PCR result in 95% of samples). The specificity of the assay was evaluated using 72 strains of V. parahaemolyticus and other bacteria. A validation of the assay with clinical samples confirmed its sensitivity and specificity. Our data suggest the newly established multiplex real-time PCR assay is practical, cost-effective, specific, sensitive and capable of high-throughput detection of pathogenic V. parahaemolyticus., (Copyright © 2014. Published by Elsevier Ltd.)

Serum response factor (SRF) function is essential for transcriptional regulation of numerous growth-factor-inducible genes and triggers proliferation, differentiation and apoptosis of the cells. In this report, the first mollusk serum response factor like homolog gene (designated ChSRF) was identified and characterized from the Hong Kong oyster, Crassostrea hongkongensis. The full-length cDNA of ChSRF was 1716 bp in length and encodes a putative protein of 434 amino acids respectively, and shares the MADS domain at the N-terminal. ChSRF is ubiquitously expressed in various tissues, with the highest expression level observed in muscle. Temporal expression of ChSRF following microbe infection shows that the expression of ChSRF in hemocytes increases from 3 to 24 h post-challenge. As a target gene of SRF, β-actin demonstrates a similar gene expression mode in constitutive tissue and pathogen infection. Furthermore, some protein profiles of ChSRF was revealed, fluorescence microscopy results show that ChSRF located in the nuclei of HeLa cells and over-expression of ChSRF activated the transcriptional activities of MAPK signal pathway in HEK293T cells. These results indicate that ChSRF maybe play an important role in signal transduction in the immunity and development response of oysters. Furthermore, we found that ChSRF could regulate the expression of β-actin gene, which indicate that ChSRF is a muscle differentiation regulator in the oyster and it will help us to improve aquaculture production., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this study, vegetative cell suspensions of two Bacillus subtilis strains, L10 and G1 in equal proportions, was administered at two different doses 10(5) (BM5) and 10(8) (BM8) CFU ml(-1) in the rearing water of shrimp (Litopenaeus vannamei) for eight weeks. Both probiotic groups showed a significant reduction of ammonia, nitrite and nitrate ions under in vitro and in vivo conditions. In comparison to untreated control group, final weight, weight gain, specific growth rate (SGR), food conversion ratio (FCR) and digestive enzymatic activity were significantly greater in the BM5 and BM8 groups. Significant differences for survival were recorded in the BM8 group as compared to the control. Eight weeks after the start of experiment, shrimp were challenged with Vibrio harveyi. Statistical analysis revealed significant differences in shrimp survival between probiotic and control groups. Cumulative mortality of the control group was 80%, whereas cumulative mortality of the shrimp that had been given probiotics was 36.7% with MB8 and 50% with MB5. Subsequently, real-time RT-PCR was employed to determine the mRNA levels of prophenoloxidase (proPO), peroxinectin (PE), lipopolysaccharide- and β-1,3-glucan- binding protein (LGBP) and serine protein (SP). The expression of all immune-related genes studied was only significantly up-regulated in the BM5 group compared to the BM8 and control groups. These results suggest that administration of B. subtilis strains in the rearing water confers beneficial effects for shrimp aquaculture, considering water quality, growth performance, digestive enzymatic activity, immune response and disease resistance., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Heat stress and pathogen infection have been considered as the main causes for mass mortality of cultured scallops during summer. In the present study, the expression profiles of proteins in the hepatopancreas of scallop Chlamys farreri were examined to reveal the possible mechanisms of physiological responses of scallop beneath heat stress and bacterial infection. An earlier occurred and higher mortality was observed in the scallops from combination treated group (28 °C and an injection of Vibrio anguillarum) in comparison to those in heat stress (28 °C) and bacteria challenge (V. anguillarum injection only) group, as well as control (PBS) and blank (untreated) group. The proteins in the hepatopancreas from scallops post 6 h of treatment were analyzed by using 2-D PAGE and ImageMaster 2D Platinum. There were total 1003 spots detected in control group, 1193 spots in heat stress group, 1263 spots in bacteria challenge group, and 1241 spots in the combination group. Fifteen protein spots expressed differentially between the combination treatment group and the bacteria challenge group were successfully identified by mass spectrometry and they were mainly classified as binding and catalytic proteins, such as endoglucanase, methylmalonate-semialdehyde dehydrogenase, xylose isomerase, tryptophanyl-tRNA synthetase, 40s ribosomal protein SA, glutathione S-transferase 4, and Mitochondrial transcription factor A, etc. These results indicated that the mortality of scallops suffered from the combination treatment was probably attributed to the impaired modulation of digestion and metabolism and ruined protein synthesis caused by heat stress together with bacteria infection. These data also provided valuable insights into the possible mechanisms of summer mortality occurrence of scallop at protein level., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Wild or farmed abalone are regularly exposed to stressors, such as air exposure and handling. Immune and transcriptional responses as well as susceptibility to vibriosis of sexually mature or immature European abalone acclimated at 16 or 19 °C were determined following handling or air exposure. Hemocyte density and H2O2 production increased while hemocyte viability and phagocytic index decreased following handling. Air exposure induces a decrease of hemocyte density and phagocytic index. Measurement of the expression of genes implicated in general metabolic, immunological and stress responses in gills, foot-muscle and hemocytes by real time q-PCR suggested that both stressors lead to a metabolic rate depression, characterized by a general inhibition of transcription. Finally, following handling a Vibrio harveyi challenge enhances almost 100% mortality of sexually immature animals at 19 °C while it has been previously demonstrated that only mature are susceptible to vibriosis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Growing evidence suggests that the transcription factor activator protein-1 (AP-1), a downstream target of mitogen-activated protein kinase (MAPK) signaling, plays a major role in stimulating the synthesis of immune effector molecules during innate immune responses. We have characterized ChAP-1, an AP-1-like protein in Crassostrea hongkongensis that is a member of the AP-1 family of proteins. ChAP-1 is composed of 290 amino acid residues with a Jun and bZIP domain at the N- and C-termini, respectively, a structure similar to that of known Ap-1 proteins. ChAP-1 mRNA is expressed in several tissues analyzed, with highest expression in the mantle. Expression of ChAP-1 increases in response to Vibrio alginolyticus, Salmo haemolyticus or Salmo cerevisiae infection and, despite the location of GFP-tagged full-length ChAP-1 protein in the cytoplasm, ChAP-1 activates the transcription of an L8G5-luc reporter gene, and its over-expression can also activate the AP-1-Luc reporter gene in HEK293T cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

As a member of the interleukin (IL)-1 family, IL-1β is a prototypical proinflammatory cytokine, which plays a crucial role in immune responses. Herein, we reported the full length cDNA of IL-1β in half-smooth tongue sole (Cynoglossus semilaevis). The csIL-1β cDNA contained a 130 bp 5' UTR, a 417 bp 3' UTR and a 741 bp coding sequence (CDS) that translated into a polypeptide of 246 amino acids. The protein sequence included a typical IL-1 family signature and lacked an interleukin-converting enzyme (ICE) cut site. RT-PCR analysis indicated a broad expression of csIL-1β, especially in immune-related organs. After injection with inactive Vibrio anguillarum, the expression of csIL-1β was induced in the head kidney and spleen and reached the highest level at 8 h post injection. Higher expression of csIL-1β was observed at gastrula stage, eye-bud stage and hatching stage and lower level of csIL-1β mRNA was detected at metamorphic stage. The expression of csIL-1β during development suggested IL-1β might be involved not only in immunity but also development. Taken together, the present study indicated that csIL-1β played an important role in immune responses and development like its mammalian counterparts, although species-specific features were present., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Sialic acid binding lectin (SABL) is a member of immunoglobulin-like lectins family that are thought to promote cell-cell interactions and regulate the functions of cells in the innate and adaptive immune systems through glycan recognition. In the present study, the full-length cDNA of SABL was identified from Manila clam Venerupis philippinarum (denoted as VpSABL) by cDNA library and RACE approaches. The cDNA of VpSABL consisted of a 5'terminal untranslated region (UTR) of 62 bp, a 3' UTR of 354 bp with a poly (A) tail, and an open reading frame (ORF) of 588 bp encoding a polypeptide of 195 amino acids with a typical C1q domain in the C-terminus. Multiple alignment analysis indicated that the deduced amino acid of VpSABL shared higher positive to other SABLs and C1q-contained proteins and should be adopted typical 10 β-strand jelly-roll folding topology common to all C1q-TNF family. Spatial expression analysis indicated that mRNA transcript of VpSABL was predominantly detectable in tissues of mantle, hepatopancreas and gill, and to a lesser degree in the tissues of muscle and haemocytes. After challenged by Vibrio anguillarum, the mRNA level of VpSABL in overall haemocytes population was recorded by quantitative real-time RT-PCR. VpSABL mRNA was down-regulated in the first 24 h post-infection. Then, the expression level increased to the peak at 72 h and recovered to the original level at 96 h. All these results indicated that VpSABL was involved in the immune response against microbe infection and might be contributed to the recognition of bacterial pathogens., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Foodborne diseases and other enteric infections often occur as outbreaks and cause morbidity and mortality all over the world. In Perú, they represent a serious public health problem, and are caused by a great variety of infectious agents. For epidemiological research, a wide array of typification methods are used. One of the most important tools for the molecular subtyping of bacterial pathogens is the Pulsed Field Gel Electrophoresis (PFGE), which is a highly precise method that allows the discrimination between different bacterial isolates which are epidemiologically related. The Instituto Nacional de Salud del Perú (INS) is part of the WHO Global Foodborne Infections Network (WHO-GFN) and of the PulseNet Latin American and Caribbean Net (PN-AL & C), with whom it shares the genetic profiles of the isolated pathogenic strains, so that it is possible to compare de genotypes of similar strains found in different countries and to identify the occurrence of epidemic outbreaks in the region, strengthening the regional system of epidemiological surveillance and generating a rapid, coordinated response between the countries. We present the two last years experience including the advances in the use of these strategic tools that have allowed us to characterize genotype patterns implicated in foodborne diseases from isolates recovered in the laboratory network of Peru.

Haliotis tuberculata mortality outbreaks have occurred in France since 1998 and were attributed to a pathogenic Vibrio harveyi. These mortalities were recorded in September, a month with abalone reproduction and characterised by high seawater temperatures. The importance of gonadal maturation and temperature increase on abalone immunity and susceptibility to V. harveyi infection needed to be clarified. Therefore, an immune survey analyzing a large panel of parameters was performed from June to September 2007 on abalone from the Bay of Brest. The data obtained were put in relation with abalone reproductive status and its susceptibility to V. harveyi. Most parameters showed clear patterns from early to late summer and during gametogenesis, phagocytosis and phenoloxidase activity were reduced, whereas basal reactive oxygen species production and agglutination titres were significantly increased. Total haemocyte counts went up after the partial spawning event at the end of June, and cell complexity diminished. Using a Principal Component Analysis, the "haemolymph profile" was shown to decrease in parallel with spawning and gonadal maturation processes, and reached a minimum just after total spawning. A significant correlation between this "haemolymph profile" and disease susceptibility allowed us to establish for the first time in abalone, a clear concordance between maturation and spawning processes, immune status and abalone susceptibility to V. harveyi.

Vibrio vulnificus produces human disease associated with raw-oyster consumption or wound infections, but fatalities are limited to persons with chronic underlying illness. Capsular polysaccharide (CPS) is required for virulence, and CPS expression correlates with opaque (Op) colonies that show "phase variation" to avirulent translucent (Tr) phenotypes with reduced CPS. The results discussed here confirmed homology of a V. vulnificus CPS locus to the group 1 CPS operon in Escherichia coli. However, two distinct V. vulnificus genotypes or alleles were associated with the operon, and they diverged at sequences encoding hypothetical proteins and also at unique, intergenic repetitive DNA elements. Phase variation was examined under conditions that promoted high-frequency transition of Op to Tr forms. Recovery of Tr isolates in these experiments showed multiple genotypes, which were designated TR1, TR2, and TR3: CPS operons of TR1 isolates were identical to the Op parent, and cells remained phase variable but expressed reduced CPS. TR2 and TR3 showed deletion mutations in one (wzb) or multiple genes, respectively, and deletion mutants were acapsular and locked in the Tr phase. Complementation in trans restored the Op phenotype in strains with the wzb deletion mutation. Allelic variation in repetitive elements determined the locations, rates, and extents of deletion mutations. Thus, different mechanisms are responsible for reversible phase variation in CPS expression versus genetic deletions in the CPS operon of V. vulnificus. Repetitive-element-mediated deletion mutations were highly conserved within the species and are likely to promote survival in estuarine environments.

Considerable progress has been made in the field of invertebrate immunity through the characterization of genes involved in the response to infection and/or stress. However, the mechanisms by which commercially important marine invertebrates can successfully survive an infection remain largely unknown. For the first time in an invertebrate model, we have searched to discover genes involved in the survival capacity of shrimp using the highly pathogenic bacteria, Vibrio penaeicida. In the present study, we applied the technique of suppression subtractive hybridization (SSH) to hemocyte cDNAs from infected and uninfected shrimp, only using samples from individuals that had survived 96 h postinfection. The resulting library contains 260 expressed sequence tagged (EST) cDNA clones potentially representing highly expressed genes in surviving shrimp. Sequence similarity comparisons were made, and putative identities were assigned to clones that were at least 51% identical to known genes. This analysis showed two functional categories that were highly represented: those of genes involved in immune reactions (10.7% of the ESTs) and those involved in proliferation-hematopoiesis (10.3%). Expression pattern profile analyses of selected ESTs at different times postinfection confirmed the differential expression of the genes and efficiency of the SSH method. Differences in gene transcript abundance, for select ESTs encoding antimicrobial effectors, were evidenced by real-time PCR between shrimp that survived acute Vibrio infection and those individuals that did not survive acute Vibrio infection. These results suggest there are basic differences at the level of transcript abundance for genes directly involved in immune and hematopoietic processes from shrimp that survive and do not survive infection.

Vibrio vulnificus is an estuarine bacterium which is the causative agent of both food-borne disease and wound infection. Although V. vulnificus is commonly found in molluscan shellfish at high numbers, the incidence of disease is relatively low, leading to the hypothesis that not all strains of V. vulnificus are equally virulent. Unfortunately, there is currently no easy test to identify virulent strains of this species. We have previously identified a 200 bp randomly amplified polymorphic DNA (RAPD) PCR amplicon associated with clinical isolates. DNA sequence data from this locus in six clinical and four environmental isolates showed that the strains could be divided into two groups, which we termed C-type (correlates with clinical origin) and E-type (correlates with environmental origin). We designed PCR primers that could distinguish between the two groups, and typed 55 randomly selected strains. We found that 90% of the C-type strains were clinical isolates, while 93% of environmental isolates were classified as E-type. The region directly downstream of this locus contained a heptanucleotide sequence repeated various times depending on the strain. Using a PCR-based assay to detect the repeat number present in a given strain, we found a statistically significant correlation with the C/E type classification and the number of repeats. The data reported here are consistent with the existence of two genotypes of V. vulnificus, with the C-type being a strong indicator of potential virulence.

A total of 54 Vibrio parahaemolyticus strains including pandemic O3:K6 strains and newly emerged O4:K68, O1:K25, O1:K26, and O1:K untypeable strains (collectively referred to as the "pandemic group") were examined for their pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) profiles and for the presence or absence of genetic marker DNA sequences, toxRS/new or orf8, that had been reported elsewhere to be specific for the pandemic group. Both PFGE and AP-PCR analyses indicated that all strains of the pandemic group formed a distinct genotypic cluster, suggesting that they originated from the same clone. In addition to the pandemic group, four O3:K6 strains that did not possess the thermostable direct hemolysin (tdh) gene also belonged to this cluster and possessed the toxRS/new sequence. However, three O3:K6 strains that clearly belonged to the pandemic group by PFGE and AP-PCR did not possess the orf8 sequence. The evidence suggests that neither the toxRS/new nor the orf8 sequence is a reliable gene marker for definite identification of the pandemic group. We therefore developed a novel multiplex PCR assay specific for the pandemic group. The assay successfully distinguished pandemic group strains from other V. parahaemolyticus strains by yielding two distinct PCR products for tdh (263 bp) and the toxRS/new sequence (651 bp).

Vibriosis is an economically important disease of fish, marine invertebrates (particularly penaeid shrimps), and large marine mammals and is responsible for high mortality rates in aquaculture worldwide. Some Vibrio species are also responsible for zoonoses, whereas others are relatively nonpathogenic. Using 16S- and 23S-based PCR reactions, we obtained species-specific patterns and a 470-bp band, respectively. DNA sequences obtained on the 23S rRNA gene allowed us to identify species-specific probes for Vibrio parahaemolyticus, V. alginolyticus, V. anguillarum and for a cluster of taxonomically related species: V. carchariae/harveyi/campbelii. A phylogenetic tree based on the 23S sequences confirmed previous results obtained by Western blotting.

Isolation of Vibrio hollisae strains, particularly from the environment, is rare. This may be due, in part, to the difficulty encountered when using conventional biochemical tests to identify the microorganism. In this study, we evaluated whether two particular genes may be useful for the identification of V. hollisae. The two genes are presumed to be conserved among the bacterial species (gyrB) or among the species of the genus Vibrio (toxR). A portion of the gyrB sequence of V. hollisae was cloned by PCR using a set of degenerate primers. The sequence showed 80% identity with the corresponding Vibrio parahaemolyticus gyrB sequence. The toxR gene of V. hollisae was cloned utilizing a htpG gene probe derived from the V. parahaemolyticus htpG gene, which is known to be linked to the toxR gene in V. hollisae. The coding sequence of the cloned V. hollisae toxR gene had 59% identity with the V. parahaemolyticus toxR coding sequence. The results of DNA colony hybridization tests using the DNA probes derived from the two genes of V. hollisae indicated that these gene sequences could be utilized for differentiation of V. hollisae from other Vibrio species and from microorganisms found in marine fish. PCR methods targeting the two gene sequences were established. Both PCR methods were shown to specifically detect the respective target sequences of V. hollisae but not other organisms. A strain of V. hollisae added at a concentration of 1 to 10(2) CFU/ml to alkaline peptone water containing a seafood sample could be detected by a 4-h enrichment incubation in alkaline peptone water at 37 degrees C followed by quick DNA extraction with an extraction kit and 35-cycle PCR specific for the V. hollisae toxR gene. We conclude that screening of seafood samples by this 35-cycle, V. hollisae toxR-specific PCR, followed by isolation on a differential medium and identification by the above htpG- and toxR-targeted PCR methods, can be useful for isolation from the environment and identification of V. hollisae.

Chemical and serological studies have been carried out on the O-antigenic lipopolysaccharides (LPS) of six strains, U-6443, W-90144, X-3972, AD-7999, 90A-6611 and KX-V212, of Vibrio parahaemolyticus isolated from patients. The O-serotypes of these strains have not been identified because they were not agglutinated by any diagnostic antisera against known O-serotype strains. A compositional sugar analysis of their LPS revealed that out of the six O-untypeable (OUT) strains, U-6443, W-90144 and AD-7999 strains belonged to chemotype II (chemotype of O2), 90A-6611 and KX-V212 strains to chemotype III (chemotype of O3, O5, O11 and O13) and X-3972 strain to chemotype IV (chemotype of O4). A structural analysis of LPS isolated from KX-V212 revealed that the inner core region of the LPS consisted of only one mole of 2-keto-3-deoxy-D-manno-octonic acid, which carried a phosphate group at position C4 and the outer core at position C5. In passive hemolysis tests performed by using LPS as the antigen to sensitize sheep red blood cells (SRBC), and diagnostic antisera (O1 to O11) or anti-whole-cell rabbit antisera raised against O12, O13 and the six OUT strains, strong cross-reactivity was observed among LPS derived from the strains belonging to chemotype II (U-6443, W-90144, AD-7999 and O2). Strong cross-reactivity was also observed between X-3972 (chemotype IV) and O4 LPS. In contrast, LPS from two of the strains belonging to chemotype III (90A-6611 and KX-V212) did not react with any of the antisera raised against known O-serotypes. Cross-absorption tests showed that the O-antigens of U-6443, W-90144 and AD-7999 were identical to that of O2, and the O-antigen of X-3972 to that of O4. On the other hand, after the absorption of antisera raised against 90A-6611 and KX-V212 with O2 cells, the hemolytic activities against SRBC sensitized with homologous LPS were still retained at a high titer, whereas the hemolytic activities against SRBC sensitized with LPS from other O-serotype strains were completely eliminated. A cross-absorption test revealed that the O-antigens of these two strains were identical to each other. Thus, it was demonstrated that the O-serotype of OUT strains 90A-6611 and KX-V212 was not involved in the known O-serotypes; rather it represented a novel serotype which has not hitherto been reported.

In order to understand the distribution and pathogenicity of vibrio in the coastal areas of China a study consisted of 51 water samples and foodstuff from 10 different areas in 5 provinces was conducted microbiologically and etiologically. Results showed that 45 pathogenic vibrio were identified, with majority of A. caviae (26.7%), V. alginolyticus (22.2%) and V. parahaemolyticus (11.1%), same kinds of vibrio found in feces from patients with acute infectious diarrhea. Prevalence rates of pathogenic vibrio identified from the East China Sea, the South China Sea and the Yellow Sea were between 66%-77%. The detection rate of pathogenic vibrio in 29 water samples was 93.1% (27/29) with 38 strains isolated mainly in river water (57.8%), followed by in sea water (21.1%) and pool water (21.1%). The results revealed that the distribution of pathogenic vibrio in different waters was closely related to its pathogenecity, providing evidence that vibrio diarrhea be supervised, diagnosed and treated in order to decrease the incidence of the disease.