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To better understand the antibiotic resistance, virulence genes, and some related drug-resistance genes of Vibrio parahaemolyticus in farmed pacific white shrimp (Litopenaeus vannamei) in Ningde regions, Fujian province, we collected and isolated a total of 102 strains of V. parahaemolyticus from farmed pacific white shrimp in three different areas of Ningde in 2022. The Kirby–Bauer disk method was used to detect V. parahaemolyticus resistance to 22 antibiotics, and resistant genes (such as quinolones (qnrVC136, qnrVC457, qnrA), tetracyclines (tet A, tetM, tetB), sulfonamides (sulI, sulII, sulIII), aminoglycosides (strA, strB), phenicols (cat, optrA, floR, cfr), β-lactams (carB), and macrolides (erm)) were detected by using PCR. The findings in this study revealed that V. parahaemolyticus was most resistant to sulfamoxazole, rifampicin, and erythromycin, with resistance rates of 56.9%, 36.3%, and 33.3%, respectively. Flufenicol, chloramphenicol, and ofloxacin susceptibility rates were 97.1%, 94.1%, and 92.2%, respectively. In all, 46% of the bacteria tested positive for multi-drug resistance. The virulence gene test revealed that all bacteria lacked the tdh and trh genes. Furthermore, 91.84% and 52.04% of the isolates were largely mediated by cat and sulII, respectively, with less than 5% resistance to aminoglycosides and macrolides. There was a clear mismatch between the antimicrobial resistance phenotypes and genotypes, indicating the complexities of V. parahaemolyticus resistance. [ABSTRACT FROM AUTHOR]

ObjectiveTo prepare proficiency testing samples of Vibrio parahemolyticus （V. parahemolyticus）and apply to laboratory test.MethodsThe five background strains and V. parahemolyticus were identified by biochemical reaction and matrix-assisted laser desorption ionization time of flight mass spectrometry（MALDI-TOF MS）. The V. parahemolyticus positive samples and negative samples were prepared by freeze-drying. The negative samples contained background bacteria only and the V. parahemolyticus were added in positive samples with background strains. Twenty samples were selected randomly for uniformity test. The samples were stored respectively at -20 ℃ and 4 ℃ to evaluate the storage stability， meanwhile the transport stability were evaluated by storing samples at 25 ℃ and 37 ℃ for 7 d. Samples were distributed to laboratories participating in the proficiency test with operation instruction， and the feedback results from laboratories were analyzed.ResultsThe results of all the related strains were consistent with the expectations. Both of the positive samples and negative samples could meet the requirements of the uniformity and stabilit. The satisfaction rates of assessment results were 87.5% and 90% in 2019 and 2020 respectively.ConclusionThe V. parahemolyticus samples could meet the requirements of the proficiency test. By organization proficiency test， the difference of laboratories can be discovered which is helpful to further improve the laboratory test capacity.

To better understand the antibiotic resistance, virulence genes, and some related drug-resistance genes of Vibrio parahaemolyticus in farmed pacific white shrimp (Litopenaeus vannamei) in Ningde regions, Fujian province, we collected and isolated a total of 102 strains of V. parahaemolyticus from farmed pacific white shrimp in three different areas of Ningde in 2022. The Kirby–Bauer disk method was used to detect V. parahaemolyticus resistance to 22 antibiotics, and resistant genes (such as quinolones (qnrVC136, qnrVC457, qnrA), tetracyclines (tet A, tetM, tetB), sulfonamides (sulI, sulII, sulIII), aminoglycosides (strA, strB), phenicols (cat, optrA, floR, cfr), β-lactams (carB), and macrolides (erm)) were detected by using PCR. The findings in this study revealed that V. parahaemolyticus was most resistant to sulfamoxazole, rifampicin, and erythromycin, with resistance rates of 56.9%, 36.3%, and 33.3%, respectively. Flufenicol, chloramphenicol, and ofloxacin susceptibility rates were 97.1%, 94.1%, and 92.2%, respectively. In all, 46% of the bacteria tested positive for multi-drug resistance. The virulence gene test revealed that all bacteria lacked the tdh and trh genes. Furthermore, 91.84% and 52.04% of the isolates were largely mediated by cat and sulII, respectively, with less than 5% resistance to aminoglycosides and macrolides. There was a clear mismatch between the antimicrobial resistance phenotypes and genotypes, indicating the complexities of V. parahaemolyticus resistance.

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Crassostrea hongkongensis (C. hongkongensis) is an important economic shellfish in marine aquaculture, but suffers from severe infection of Vibrio parahaemolyticus (V. Parahemolyticus). The study on immune strategy against Vibrio infection will help to enhance the disease resistance of C. hongkongensis. In this study, after V. parahaemolyticus challenge, the immune enzyme activities and transcriptome were analyzed to explain the immune strategy of C. hongkongensis. The immune enzyme activities analysis showed that all of superoxide dismutase (SOD), catalase (CAT), lysozyme (LZM) activities and malondialdehyde (MDA) content were significantly increased among 3 and 12 h post-infection, indicating that V. parahaemolyticus had stimulated the immune response of C. hongkongensis. Transcriptome analysis showed that a total of 636,295,318 clean readings were generated from the libraries of control and infection groups. Comparing with the control groups (C0h), 147 differentially expressed genes (DEGs) were identified in T3h group, 392 DEGs in T6h group, 47 DEGs in T12h group, and 423 DEGs in T24h group. The time-series expression profiles of DEGs showed that the rising and failing trends were significantly enriched in 2 clusters, including 158 DEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that immune-related DEGs were annotated into 20 categories, including Toll and Imd signaling pathway, IL-17 signaling pathway, Toll like receptor (TLR) pathway and c-type lectin receptor signaling pathway. Since multiple immune-related DEGs were enriched in TLR signal pathway, qRT-PCR was employed to validate the expression of genes in TLR pathway. The qRT-PCR results showed that all of six detected genes were significantly upregulated, including TLR4, MyD88, IRAK4, TRAF6, IL17 and COX2. The data indicated that TLR4-MyD88 pathway took part in the immune response of C. hongkongensis against Vibrio infection.

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

As a common aquatic pathogen, Vibrio parahaemolyticus can cause a variety of diseases of shrimp, especially acute hepatopancreatic necrosis disease (AHPND), which leads to great losses to the aquaculture industry around the world. However, the molecular mechanism of V. parahaemolyticus infection is still unclear. Neocaridina denticulate sinensis is a kind of small ornamental shrimp that is popular in aquarium trade, and due to its tenacious vitality, rapid growth, high reproductive capacity, it is very suitable to be developed as an animal model for basic research on decapod crustaceans. Thus, in this paper, transcriptomes of N. denticulate sinensis hepatopancreas with or without V. parahaemolyticus injection were explored. The results showed that a total of 23,624 genes with the N50 of 2705 bp were obtained. Comparative transcriptomic analysis revealed 21,464 differentially expressed genes between the V. parahaemolyticus infected and non-infected group, of which, 11,127 genes were up-regulated and 10,337 genes were down-regulated. Functional enrichment analysis suggested that many DEGs enriched in immune related pathways, including MAPK signaling pathway, Phosphatidylinositol signaling system, Chemokine signaling pathway, Phagosome and Jak-STAT signaling pathway and so on. Eight genes were selected randomly for qRT-PCR to verify the transcriptome sequencing results and the results showed the expression of these genes were consistent with the transcriptome results. Our work provides a unique and important dataset that contributes to the understanding of the molecular mechanisms of the immune response to V. parahaemolyticus infection and may further provide the basis for the prevention and resolution of bacterial diseases. • Transcriptomic analysis of hepatopancreas from Neocaridina denticulate sinensis challenged with Vibrio parahemolyticus. • A total of 21,464 DEGs were identified, including 11127 up-regulated genes and 10337 down-regulated genes. • Many DEGs were enriched in immune-related pathways, and expression of some PRRs and immune related factors were also altered. • Expression tendencies of 8 randomly selected genes were consistent with the result from transcriptomic analysis. [ABSTRACT FROM AUTHOR]

Objective To study the epidemiologic and etiologic characteristics of Salmonella, Shigella, Vibrio parahemolyticus, diarrheagenic Escherichia coli (DEC) from foodborne diarrhea patients in Baiyin. Methods Stool samples were collected from foodborne disease patients in Baiyin during 2016-2018 and pathogenic bacteria strains were isolated and identified by biochemical characteristics, serotyping, drug resistance test and pulsed-field gel electrophoresis (PFGE). Results A total of 300 strains were isolated from 1 003 stool samples, the overall positive rate was 29.9% and the positive rate of DEC was the highest (29.1%, 292/1 003), followed by that of Salmonella and Shigella, and Vibrio parahemolyticus were not detected. Totally 1 003 foodborne disease cases were reported and the ratio of male and female was 1.3:1, the detection rate of pathogenic bacteria in female patients (31.8%, 136/428) was higher than male patients (28.5%, 164/575), the patients were mainly adults aged 19-59 years and young children under 5 years old, and the positive rate was 31.5% (76/241) and 30.6% (188/615). Pathogenic bacteria strains were isolated mainly in the second and third quarter of the year. PFGE result indicated that 7 isolates belonged to 6 types, and there was one aggregation phenomenon. Salmonella had high resistant rate to tetracycline (85.7%, 6/7), it was found that 6 strains were multi-drug resistant. Shigella flexneri 2a subtype was resistant to 7 drug.292 strains of DEC were sensitive to imipenem, and the drug sensitive rates of ceftazidime, cefoxitin, chloramphenicol and ciprofloxacin were greater than 80%, the resistance rate to ampicillin was the highest (80.8%, 236/292), the multi-resistance to three or more antibiotics was 75.3% (220/292). Conclusion In the active etiological surveillance for foodborne diseases in Baiyin during 2016-2018, the detection rate of DEC was the highest. Pathogenic bacteria strains were isolated mainly in adults aged 19-59 years. The season specific characteristics of pathogenic bacteria was observed, and the infections mainly occurred in the second and third season. Multidrug resistance of pathogenic bacteria strains was serious with broad-spectrum.

[Objectives] The structural components and the biological functions of Vibrio parahemolyticus T3SS1 have not been fully described. In this study we investigated the role of gene vscI. [Methods] To do some research about the vscI,we constructed the deletion strain ΔvscI and complementation strain CΔvscI to analyze the cytotoxicity,adhesion and other biological characteristics of wild strain(POR-1),ΔvscI and CΔvscI when infected HeLa. Further,Western blot analysis and ELISA kits were used to research the translocation of effector protein in Hela cell. [Results] Compared with POR-1 infection group,the results showed that the number of apoptosis decreased significantly by about 80%(P<0.01),the adhesion decreased significantly by 85%(P<0.01),and the cytotoxicity decreased significantly by 60%(P<0.01) in ΔvscI infection group. In addition,Western blot and ELISA data indicated that the intracellular cAMP levels of the ΔvscI group was significantly lower than those of the POR-1,CΔvscI. [Conclusions] vscI improves the adhesion ability of V.parahaemolyticus to cells,and produces cytotoxicity,promotes cell apoptosis,and it is necessary for the translocation of T3SS1 effector. [ABSTRACT FROM AUTHOR]

In farmed saltwater fish, vaccination is considered the first barrier against pathogenic agents. In Egypt, the vaccine application can diminish the dilemma of summer mass kills of farmed marine fish. The efficacy of booster vaccination with a bivalent vaccine against vibriosis was studied in European seabass. Fish were vaccinated by direct immersion (DI) in water containing bivalent Vibrio alginolyticus and Vibrio parahemolyticus whole cells killed vaccine. Immersion vaccination is more applicable under semi‐intensive fish culture, but less potent than injection. Accordingly, to increase antigen uptake and conferred protection, vaccinated fish randomly received booster vaccination via direct immersion (DI imm.) or intraperitoneal injection (i.p.) at 21 days post‐prime vaccination (21 dpv). Vaccinated and unvaccinated fish were monitored for 3 months and challenged with V. alginolyticus and/or V. parahemolyticus at 42 dpv. Specific and non‐specific immune responses were evaluated in blood samples. At 52 dpv (10 days post‐challenge), the relative immunoglobulin T (IgT) expression was assessed in the gills, hindgut, head kidney and spleen. Notably, imm. + imm. and imm. + i.p. vaccination yielded significantly higher specific antibody titre at 70 dpv, whereas the former was associated with significantly higher IgT expression in the gills and hindgut (p <.05) than the latter. High IgT levels in mucosal organs (gills and hindgut) might be the first indicator of the involvement of IgT in mucosal immune responses. Relative per cent survival among booster‐vaccinated fish ranged from 70% to 90%. The results support the use of this vaccination protocol as a highly effective tool to protect seabass from vibriosis and to improve fish health and environment. Booster doses are strongly recommended under farming environments. [ABSTRACT FROM AUTHOR]

Thymol has efficient bactericidal activity against a variety of pathogenic bacteria, but the bactericidal mechanism against Vibrio parahemolyticus ( V. parahemolyticus ) has rarely been reported. In the current study, we investigated the bactericidal mechanism of thymol against V. parahemolyticus . The Results revealed that 150 μg/mL of thymol had 99.9% bactericidal activity on V. parahemolyticus . Intracellular bursts of reactive oxygen species (ROS), Fe 2+ accumulation, lipid peroxidation, and DNA breakage were checked by cell staining. The exogenous addition of H 2 O 2 and catalase promoted and alleviated thymol-induced cell death to a certain extent, respectively, and the addition of the ferroptosis inhibitor Liproxstatin-1 also alleviated thymol-induced cell death, confirming that thymol induced Fenton-reaction-dependent ferroptosis in V. parahemolyticus . Proteomic analysis revealed that relevant proteins involved in ROS production, lipid peroxidation accumulation, and DNA repair were significantly upregulated after thymol treatment. Molecular docking revealed two potential binding sites (amino acids 46H and 42F) between thymol and ferritin, and thymol could promote the release of Fe 2+ from ferritin proteins through in vitro interactions analyzed. Therefore, we hypothesized that ferritin as a potential target may mediate thymol-induced ferroptosis in V. parahemolyticus . This study provides new ideas for the development of natural inhibitors for controlling V. parahemolyticus in aquatic products.

Fresh foods are vulnerable to foodborne pathogens which cause foodborne illness and endanger people’s life and safety. The rapid detection of foodborne pathogens is crucial for food safety surveillance. An in situ-synthesized gene chip for the detection of foodborne pathogens on fresh-cut fruits and vegetables was developed. The target genes were identified and screened by comparing the specific sequences of Salmonella Typhimurium, Vibrio parahemolyticus, Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli O157:H7 from the National Center for Biotechnology Information database. Tiling array probes were designed to target selected genes in an optimized hybridization system. A total of 141 specific probes were selected from 3,227 hybridization probes, comprising 26 L. monocytogenes, 24 S. aureus, 25 E. coli O157:H7, 20 Salmonella Typhimurium, and 46 V. parahemolyticus probes that are unique to this study. The optimized assay had strong amplification signals and high accuracy. The detection limit for the five target pathogens on fresh-cut cantaloupe and lettuce was approximately 3 log cfu/g without culturing and with a detection time of 24 h. The detection technology established in this study can rapidly detect and monitor the foodborne pathogens on fresh-cut fruits and vegetables throughout the logistical distribution chain, i.e., processing, cleaning, fresh-cutting, packaging, storage, transport, and sale, and represents a valuable technology that support the safety of fresh agricultural products.

Abstract Background Acute hepatopancreatic necrosis disease (AHPND) has emerged as a major debilitating disease that causes massive shrimp death resulting in substantial economic losses in shrimp aquaculture. Given that several diseases and infections have been associated with microRNAs (miRNAs), we conducted a comparative transcriptomic analysis using the AHPND (VA) and non-AHPND (VN) strains of Vibrio parahemolyticus to identify miRNAs potentially involved in AHPND pathogenesis in Litopenaeus vannamei. Results A total of 83 miRNAs (47 upregulated and 36 downregulated) were significantly differentially expressed between the VA and VN challenged groups, while 222 target genes of these miRNAs were predicted. Functional enrichment analysis revealed that the miRNAs target genes were involved in multiple biological processes including metabolic pathways, amoebiasis, Vibrio cholerae infection etc. Finally, interaction network and qPCR (Real-time Quantitative PCR) analysis of 12 potential key AHPND-related miRNAs and their predicted target genes, revealed their possible involvement in modulating several immune-related processes in the pathogenesis of AHPND. Conclusions We have shown using comparative transcriptomic analysis, miRNAs and their target genes that are responsive to AHPND V. parahemolyticus infection in shrimp, therefore suggesting their possible role in defense response to AHPND V. parahemolyticus infection.

Objectives: In this study, bioengineered AgNPs (silver nanoparticles) from Spirulina platensis was used to counter the biofilm mediated virulence by Vibrio parahemolyticus. Methods: AgNPs were synthesized from crude Spirulina platensis extract and characterized using UV spectrophotometry, Transmission electron microscope (TEM). Antibacterial effect against aquatic pathogen Vibrio parahemolyticus was carried out. Further, the nanoparticle at sub MIC (minimum inhibitory concentration) level were screened for anti-virulence property under in vitro studies which include biofilm inhibition using microtiter plate assay, microscopic analysis under light microscope and SEM (Scanning electron microscope) imaging, EPS (Exopolysaccharide) inhibition, CSH(Cell surface hydrophobicity) inhibition using BATH(Bacterial adhesion to hydrocarbons) assay, swarming and swimming motility inhibition, growth pattern analysis followed by in vivo study in aquatic animal model brine shrimp and its survival percentage along with light microscopic imaging was performed. Results: AgNPs, at 320µg/ml showed bactericidal activity, minimal inhibitory concentration was found to be 160 µg/ml. All anti-virulence assay performed at below sub MIC concentration. Biofilm and EPS showed 63% and 53% inhibition respectively, CSH was found to be decreased 3-fold. Growth pattern at sub MIC compared with non-treated showed similar result. In vivo study increased survival percentage (90%) when treated with AgNPs, compared to untreated (20%). A 7-fold increased resistance to pathogen was found and light microscope also confirms the efficacy of Ag nanoparticle. Conclusion: AgNPs become a substitute to the current commercial antibiotics used in aquaculture against Vibriosis, by functioning as an efficient quorum sensing inhibitor. [ABSTRACT FROM AUTHOR]

Interleukin-1 receptor-associated kinases (IRAKs) play important roles in the innate immune system of TLR (Toll-like receptor) signaling pathway. In this paper, interleukin-1 receptor-associated kinase-b (designated as McIRAK-b) and interleukin-1 receptor-associated kinase-a (named as McIRAK-a) were obtained based on the transcriptome data, the full length of McIRAK-b was 1815 bp and McIRAK-a was 3168bp, encoding 532 and 978 amino acids, respectively. BLASTp analysis and phylogenetic relationship strongly suggested that the deduced amino acid sequence of McIRAK-b had high homology with IRAK-4 and McIRAK-a was similar to IRAK-1 of other mollusks, especially at their function domains. The expressions of McIRAK-b and McIRAK-a were detected in six tissues including adductor muscle, hemocyte, gills, gonad and hepatopancreas, and the highest expressions appeared both in gills. The expressions of McIRAK-b and McIRAK-a in gills were observed with time-dependent manners after bacterial infections. After being challenged with Vibrio alginolyticus , McIRAK-b expressed significantly and got the peak at 8 h (9.47 times compared with the control group), but the peak appeared at 4 h by being infected with Vibrio parahaemolyticus (12.02 times compared with the control group). The highest point of McIRAK-a mRNA appeared at 12 h (5.16 times) after being challenged with V.alginolyticus and 8 h (4.21 times) for V.parahaemolyticus challenge. The results suggested that IRAK-b and IRAK-a might be important in immune signaling pathway of mussels. The kinase functional domain sequences (S_TKc) of McIRAK-b and McIRAK-a expressed in BL21(DE3) and purified by Ni-NAT Superflow resin conforming to the expected molecular weight with many active sites for their conferring protein-protein interaction functions. This study may provide some further understandings of the regulatory mechanisms in the bivalve innate immune system for IRAKs family. • IRAK-b and IRAK-a were identified from Mytilus coruscus. • The functional domains and some crucial amino acids in two IRAKs were conserved. • They were constitutively expressed in different tissues but the highest was in gill. • Their temporal expressions were up-regulated under the stress of aquatic pathogens. • S_TKc in IRAKs were expressed and purified conforming to the expected molecular weight. [ABSTRACT FROM AUTHOR]

Vibrio parahemolyticus is a halophilic bacterium which causes widespread seafood poisoning pathogenicity. Although the incidence of disease caused by V. parahemolyticus was stepwise increased, the pathogenic mechanism remained unclear. Herein, the difference of V. parahemolyticus's metabonomic which on blood agar and seawater beef extract peptone medium was detected via nuclear magnetic resonance and 55 metabolites were identified. Among them, 40 kinds of metabolites were upregulated in blood agar group, and 12 kinds were downregulated. Nine pathways were verified by enrichment analysis which were predicted involved in amino acids and protein synthesis, energy metabolism, DNA and RNA synthesis and DNA damage repair. We supposed that the metabolic pathway obtained from this study is related to V. parahemolyticus pathogenicity and our findings will aid in the identification of alternative targets or strategies to treat V. parahemolyticus-caused disease. [ABSTRACT FROM AUTHOR]

Accurate and highly sensitive detection of viable but nonculturable (VBNC) Vibrio parahaemolyticus is crucial for preventing seafood-related outbreaks. The VBNC state may occur due to bacterial susceptibility to cold shock during food storage. Propidium monoazide (PMA) has been widely applied to detect VBNC foodborne pathogens. In this study, we developed and compared real-time PCR (qPCR) and real-time LAMP (qLAMP) methods combined with an improved propidium monoazide (PMAxx) to detect pathogenic VBNC V. parahaemolyticus. The designed primers and probes were determined to be highly selective for V. parahaemolyticus strains. The combined PMAxx-qPCR method, requiring 100 min, demonstrated a quantification limit of 10.5 colony-forming units (CFU)/mL in pure culture and 28 CFU/g in raw shrimp respectively, which were 10-fold lower than the PMAxx-qLAMP method (45min). When testing mixtures containing different ratios of VBNC to dead V. parahaemolyticus , PMAxx-based methods were notably superior at distinguishing between VBNC and dead bacteria when VBNC cell concentrations were low. Therefore, PMAxx is an effective means for improving the detection and quantification of VBNC V. parahaemolyticus by qPCR and qLAMP. Pretreatment with PMAxx was shown to be suitable for detection of VBNC V. parahaemolyticus in complex food samples, including raw shrimp. • PMAxx-qPCR and PMAxx-qLAMP were developed and compared to detect VBNC Vibrio parahaemolyticus. • Both methods had good specificity, but PMAxx-qPCR had higher sensitivity. • Respective LoD in pure culture and raw shrimp were 10.5 CFU/mL and 28 CFU/g without a prior enrichment step. • PMAxx exhibited higher accuracy than PMA in quantitative detection. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) play an important role in defense response to pathogens in mollusk. In this study the first TLR from Sepiella japonica (named as Sj TLR) was functionally characterized, and its full-length cDNA consisted of 3914bp (GenBank accession no. AQY56780.1) including an open reading frame of 3582bp, encoding a putative protein of 1193 amino acids. Its theoretical molecular weight was 137.87 KDa and the predicted isoelectric point was 3.69. The derived amino acids sequence comprised of an extracellular domain including 26 amino acids signal peptide and eleven leucine-rich repeats (LRR), capped with LRRCT and LRRNT followed by transmembrane domain and cytoplasmic Toll/IL-1R domain (TIR). In addition, 12 potential N -linked glycosylation sites were present in the ectodomain to influence protein trafficking, surface presentation and ligand recognition. Multiple sequence alignment and phylogenetic analysis revealed that Sj TLR shared the highest similarity to that of Euprymna scolopes and they fell into the same clade. Real-time PCR showed Sj TLR expressed constitutively in all tested tissues, including gill, liver, brain, muscle, intestine, heart, lobus opticus and stomach, but showed different expression levels with genders. The highest expression was in the liver, and the lowest was in stomach for both genders. The functional domain region sequences encoding LRRs domain protein and TIR domain containing protein (TcpB) were expressed in BL21(DE3) respectively and purified with Ni-NAT Superflow resin conforming to the expected molecular weight. The cellular localization of Sj TLR in HEK293 cells was conducted and plasma membrane localization was detected. Sj LRRs internalization upon the activation of LPS was also observed, and dramatic redistribution of Sj LRRs in the cytoplasm with distinct perinuclear accumulation was found. After Sj TLR transfection Toll/NF-κB signaling pathway was active in HEK293 treated with LPS and TNFɑ. The nuclear related genes may also be activated by NF-κB in the nucleus, and the corresponding mRNA was transferred through the intracellular signal transduction pathway, so that IL-6 cytokines could be synthesized and released. After infection by Vibrio parahemolyticus and Aeromonas hydrophila the expression of Sj TLR were upregulated with time-dependent manner. These findings might be valuable for understanding the innate immune signaling pathways of S.japonica and enabling future studies on host-pathogen interactions. • A TLR homologue was identified from Sepiella japonica (SjTLR). • SjTLR located on membrane and SjLRRs migrated inward by the activation of LPS. • After Sj TLR transfection NF-κB signal pathway was active treated with LPS and TNFɑ. • After infection by Vibrio the expression of SjTLR were upregulated. • The results might be helpful to comprehend immune response of TLR in cephalopoda. [ABSTRACT FROM AUTHOR]

Abstract Interleukin-8 (IL-8 or chemokine (C-X-C motif) ligand 8, CXCL8) is a chemokine produced by multiple cell types. It promotes chemotaxis and phagocytosis via interaction with chemokine receptors CXCR1 and CXCR2. Using published data, IL-8 gene (LcIL-8) of the large yellow croaker (Larimichthys crocea) was cloned into the pcDNA3.1 plasmid, and an interleukin-8 receptor (LcCXCR2) was cloned into the pEGFP-N1 plasmid. Secratory expression of Lc IL-8 in HEK293T cells was carried out, and product in culture medium was collected for Lc CXCR2 stimulation in HEK293 cells. Following receptor internalization observation and intracellular signaling detection, the functional interaction of Lc IL-8 and Lc CXCR2 was further determined and the ERK phosphorylation signal activation mediated by Lc CXCR2 was demonstrated. Quantitative real-time PCR analysis was used to analyze transcription level regulation of Lc IL-8 and Lc CXCR2 in various tissues of large yellow croaker. Expression of LcIL-8 and LcCXCR2 was elevated in the spleen, head kidney, and liver after Vibrio parahemolyticus challenge. Results illustrated the functional interaction between Lc IL-8 and Lc CXCR2 in mediating intracellular ERK1/2 phosphorylation signaling and suggested that the Lc IL-8 and Lc CXCR2 system is part of the immune response induced by V. Parahemolyticus in L. crocea. Highlights • The full length CDS of Lc CXCR2 from spleen of large yellow croaker was cloned. • The cell surface localization of Lc CXCR2 was determinated. • Fusion expression of Lc IL-8 and Lc CXCR2 was conducted in HEK293T and HEK293 cells. • The functional inteaction between Lc IL-8 and Lc CXCR2 was demonstrated and their immunoregulatory function has been revealed. [ABSTRACT FROM AUTHOR]

Abstract In this study, we sequenced and characterized two homologues of interferon-stimulated gene ISG15, termed as LcISG15-1 and LcISG15-2, from the large yellow croaker (Larimichthys crocea). The LcISG15-1 encodes 159 amino acids and the LcISG15-2 encodes 155 amino acids, both of which contain two tandem ubiquitin-like domains and the conserved C-terminal LRGG conjugation motif. The sequence analyses showed that both the LcISG15-1 and LcISG15-2 exhibit high similarity with ISG15 from other fishes. A putative IFN-stimulatory response element (ISRE) was detected in promoter regions of both the LcISG15-1 and LcISG15-2. Phylogenetic analyses revealed a close evolutionary relationship of both the LcISG15-1 and LcISG15-2 with other teleostean ISG15. Molecular evolutionary analyses suggested a gene duplication event of ISG15 in the ancestor of the Sciaenidae, with a signature of positive selection was found in the ISG15-2 gene copy of sciaenid fishes. The Real-time PCR analyses showed that the LcISG15-1 and LcISG15-2 were both found to be ubiquitously expressed in ten examined organs in large yellow croaker, with predominant expressions both in peripheral blood. Expression analyses showed that both the LcISG15-1 and LcISG15-2 were rapidly and significantly upregulated in vivo after poly (I:C) challenge in liver and spleen organs. However, the LcISG15-1 and LcISG15-2 were both significantly induced after pathogen Vibrio parahemolyticus infection only in the liver but not in the spleen. These results indicated that there are two ISG15 homologues in the large yellow croaker, both of which are likely to be involved in host immune defense against viral and bacterial infection. Highlights • LcISG15-1 and LcISG15-2 were sequenced from Larimichthys crocea. • LcISG15-1 and LcISG15-2 both express ubiquitously with predominant expressions in peripheral blood. • LcISG15-1 and LcISG15-2 were significantly upregulated after poly(I:C) challenge in liver and spleen. • LcISG15-1 and LcISG15-2 were significantly upregulated after Vibrio parahemolyticus infection only in liver but not in spleen. • LcISG15-1 and LcISG15-2 both play important roles in host immune defense against viral and bacterial infection. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Food Research & Development is the property of Food Research & Development Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Armadillo repeat-containing proteins (ARMCs) comprise a large family that is widely distributed in eukaryotes and plays prominent roles in cell-cell adhesion, intracellular signaling, and cytoskeletal regulation. In this study, three ARMC genes, termed LvARMC6 , LvARMC7 and LvARMC8 , were identified and characterized from Litopenaeus vannamei. The complete cDNAs open reading frames (ORF) of LvARMC6 , LvARMC7 , and LvARMC8 (GenBank accession no. MG735126, MG728109 and KX058562) were 1410 bp, 570 bp and 2046 bp, encoding 469, 189, and 681 amino acids, respectively. Topology analysis indicated that three ARM domains were present in LvARMC6, one in LvARMC7 and six in LvARMC8. The identities of all the three LvARMCs with other species were between 50% and 71%. Phylogenetic analysis illustrated that different subtype of ARMCs formed their own separate branches and LvARMCs were placed in branch of invertebrates respectively with strong bootstrap support. The constitutive expressions of LvARMCs were confirmed by real-time quantitative PCR. LvARMC6 , LvARMC7 and LvARMC8 were expressed highest in heart, gills and epithelium, respectively. After challenge with either white spot syndrome virus (WSSV), Vibrio parahemolyticus , or Staphylococcus aureus , all of the LvARMCs demonstrated differential expression profiles in hemocytes, hepatopancreas, intestine and gills. Taken together, our results suggest that LvARMCs may play a role in the innate immune defense against pathogenic viral and bacterial infections of L. vannamei. Highlights • Three ARMC genes, named LvARMC6 , LvARMC7 and LvARMC8 , were cloned from Litopenaeus vannamei. • Diversity of protein structures among ARMCs from different species was shown by topology analysis. • Phylogenetic analysis indicated that LvARMCs were most closely related to ARMC genes from other invertebrates. • Lv ARMC s were constitutively expressed in L. vannamei. • After challenge by different pathogens, differential expression profiles of LvARMCs were demonstrated in various tissues. [ABSTRACT FROM AUTHOR]

Abstract As an important economical shellfish in coastal area of China, abalone is susceptible to bacterial infection, especially Vibiro parahemolyticus (V. parahemolyticus). Matrix metalloproteinases (MMPs) have been extensively investigated in the immune response of mammals. However, little is known about the involvement of MMP in abalone innate immune system against pathogen infection. In this study, the role of MMP-1 in the immune response of Pacific abalone (Haliotis discus hannai) was explored. The results showed that V. parahemolyticus infection induced significantly elevated expression of MMP-1 as well as immune related genes including allograft inflammatory factor 1 (AIF-1), macrophage expressed gene 1 (MPEG-1) and TPA-inducible sequence 11 family protein (Tis11FP). Notably, silencing of MMP-1 reduced the expression of these genes, suggesting that MMP-1 was an upstream regulatory factor in V. parahemolyticus infection. Further analysis showed that MMP-1 was engaged in the regulation of cellular (phagocytosis, apoptosis) and humoral [superoxide dismutase (SOD), alkaline phosphatase (ALP), acid phosphatase (ACP)] immunity. Interestingly, the extracellularly distributed MMP-1 could be translocated to the nuclei of hemocytes, thereby functioning as a transcriptional regulator or by selectively activating or inactivating other components through proteolysis. Hence, our study established an important role of MMP-1 in abalone innate immunity against V. parahemolyticus infection and it represented the first report on the investigation of MMP in abalone. Highlights • Induced expression of MMP-1 was observed after Vibrio parahemolyticus infection in Pacific abalone (Haliotis discus hannai). • MMP-1 was involved in the regulation of phagocytosis and apoptosis activities in abalone. • SOD, ALP and ACP activities were regulated by MMP-1 in abalone. • MMP-1 altered the expression of AIF-1, MPEG-1 and Tis11FP genes. • MMP-1 could be translocated into the nuclei of abalone hemocytes post bacterial infection. [ABSTRACT FROM AUTHOR]

The presence of Vibrio parahaemolyticus in oyster mass, commercialized in localities of southeastern Cuba was evaluated. The selected harvest areas were named as A, B and C. Samples were taken in March, April and May of 2017 from water; oyster mass in its shell; and; refrigerated and frozen oyster mass. Two detection test methods were used: qualitative (presence/absence); and semi-quantitative (most probable number (MPN), followed by confirmation through selected biochemical tests. The results showed a relationship between low water salinity (25 practical salinity units); and a higher concentration of total Vibrio parahaemolyticus. The chilled oyster mass was found with total number of Vibrio parahaemolyticus levels of up to 102 MPN/g. Although this value does not exceed the infection dose of oysters served, it is necessary to ensure the hygiene quality and safety of this product to minimize the potential risk of gastroenteritis. [ABSTRACT FROM AUTHOR]

Abstract Toll-like receptor ( TLR) is considered to be an evolutionarily conserved transmembrane protein which promotes the Toll signal pathway to active the expression of transcription factors in the innate immunity of the organism. In this study, a full length of TLR homologue of 2525bp in Mytilus coruscus (named as McTLR-a, GenBank accession no: KY940571) was characterized. Its ORF was 1815 bp with a 5'untranslated region (UTR) of 128 bp and a 3′UTR of 582 bp, encoding 602 amino acid residues with a calculated molecular weight of 70.870 kDa ( pI = 6.10). BLASTn analysis and phylogenetic relationship strongly suggested that this cDNA sequence was a member of TLR family. Quantitative real time RT-PCR showed that constitutive expression of McTLR-a was occurred, with increasing order in hemocyte, gonad, mantle, adducter, gill and hepatopancreas. Bacterial infection and heavy metals stimulation up-regulated the expression of McTLR-a mRNA in hepatopancreas with time-dependent manners. The maximum expression appeared at 12 h after pathogenic bacteria injection, with approximately 22-fold in Aeromonas hydrophila and 17-fold in Vibrio parahemolyticus higher than that of the blank group. In heavy metals stress group, they all reached peaks at 3d, while the diverse concentration caused the maximum expression were different. The highest expression reached approximately 7-fold higher than the blank in low concentration of Pb2+ exposure. In Cu2+ treated group, it reached the peak (approximately 12-fold higher than the blank)in middle concentration. These results indicated that McTLR-a might be involved in the defense response and had a significant role in mediating the environmental stress. Highlights • A Toll-like receptor homologue was identified from Mytilus coruscus (McTLR-a). • The functional domains in McTLR-a were conserved. • McTLR-a was constitutively expressed in different tissues but highest in hepatopancreas. • Its temporal expression was up-regulated under various stressors. • The results might be helpful to comprehend immune response of TLR in bivalve. [ABSTRACT FROM AUTHOR]

Background: Acute hepatopancreatic necrosis disease (AHPND) has emerged as a major debilitating disease that causes massive shrimp death resulting in substantial economic losses in shrimp aquaculture. Given that several diseases and infections have been associated with microRNAs (miRNAs), we conducted a comparative transcriptomic analysis using the AHPND (VA) and non-AHPND (VN) strains of Vibrio parahemolyticus to identify miRNAs potentially involved in AHPND pathogenesis in Litopenaeus vannamei. Results: A total of 83 miRNAs (47 upregulated and 36 downregulated) were significantly differentially expressed between the VA and VN challenged groups, while 222 target genes of these miRNAs were predicted. Functional enrichment analysis revealed that the miRNAs target genes were involved in multiple biological processes including metabolic pathways, amoebiasis, Vibrio cholerae infection etc. Finally, interaction network and qPCR (Real-time Quantitative PCR) analysis of 12 potential key AHPND-related miRNAs and their predicted target genes, revealed their possible involvement in modulating several immune-related processes in the pathogenesis of AHPND. Conclusions: We have shown using comparative transcriptomic analysis, miRNAs and their target genes that are responsive to AHPND V. parahemolyticus infection in shrimp, therefore suggesting their possible role in defense response to AHPND V. parahemolyticus infection. [ABSTRACT FROM AUTHOR]

Amphioxus, a basal chordate, is widely considered to be an existing proxy of the invertebrate ancestor of vertebrates, and it exhibits susceptibility to various pathogen infections and pathogenic mimic challenges. Here, in order to understand more clearly its antibacterial mechanisms, we analyzed the ribosomal RNA (rRNA)-depleted transcriptome of Chinese amphioxus ( Branchiostoma belcheri ) infected with Vibrio parahaemolyticus ( V. p. ) via next-generation deep sequencing technology (RNA-seq). We identified a total of 3214 differentially expressed genes (DEGs) by comparing V. p. -infected and control transcriptome libraries, including 2219 significantly up-regulated and 995 significantly down-regulated DEGs in V. p. -infected amphioxus. The DEGs with the top 10 most dramatic expression fold changes after V. p. infection, as well as 53 immune-related DEGs (IRDs) belonging to four primary categories of innate immunity were analyzed further. Through gene ontology (GO) and pathway enrichment analysis, DEGs were found to be primarily related to immune processes, apoptosis, catabolic and metabolic processes, binding and enzyme activity, while pathways involving bacterial infection, immune signaling, immune response, cancer, and apoptosis were overrepresented. We validated the RNA-seq results by detecting the expression levels of 10 IRDs using qRT-PCR, and we surveyed the dynamic variation in gene expression for these IRDs at 0, 6, 12, 24, and 48 h after V. p. treatment. Subsequently, according to the RNA-seq results, the presence of a primitive Toll-like receptor (TLR)-mediated antibacterial immune signaling pathway was predicted in B. belcheri . This study provides valuable information regarding antibacterial immunity for further research into the evolution of immunity in vertebrates and broadens our understanding of the innate immune response against bacterial invasion in amphioxus. [ABSTRACT FROM AUTHOR]

pH variation could cause a stress response in euryhaline penaeids, we evaluated the mortality, growth performance, osmoregulation gene expression, digestive enzyme activity, histology, and resistance against Vibrio parahemolyticus of white shrimp Litopenaeus vannamei reared under conditions of gradual changes to a low-pH environment (gradual-low pH, 6.65–8.20) or a high-pH environment (gradual-high pH, 8.20–9.81) versus a normal pH environment (8.14–8.31) during a 28-d experiment. Consequently, under gradual-high pH, the cumulative mortality rate (CMR) rose with time until 39.9% on days 28; the weight gain percentage (WGP) and length gain percentage (LGP) decreased continuously. However, under gradual-low pH, the CMR of shrimp stabilized at 6.67% during 7–28 d; the WGP and LGP decreased first and then returned to normal. These results indicated that L. vannamei displayed a moderate tolerance to gradual-low pH, compared with gradual-high pH. Under gradual-low pH, the Na + /K + -ATPase, cytoplasmic carbonic anydrase (CAc), and glycosyl-phosphatidylinositol-linked carbonic anhydrase (CAg) transcripts of shrimp increased continuously or then back to normal; the amylase, lipase, and trypsin activities decreased first and then returned to normal or increased; the hepatopancreases and midguts showed histopathological lesions first and then got remission. Thus, the major adaptation mechanism of shrimp to gradual-low pH might be its high osmoregulation ability, which made shrimp achieve a new, balanced steady-state, then promoted longer intestinal villi and recuperative hepatopancreases of shrimp with enhanced digestive enzyme activities to increase nutrient absorption after long-term exposure. Meanwhile, the enhanced resistance against V. parahemolyticus under gradual-low pH would probably inhibit disease outbreak in the shrimp farming. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Vibrio parahemolyticus ( V. parahemolyticus ) is a major pathogen for abalone, an important economical shellfish in coastal area of China. There is little known about the abalone innate immune system against pathogen infection. Clip-domain serine proteases (cSPs) are increasingly recognized to play important roles in host immune defense in invertebrates. In this study, we cloned a cSP (Hdh-cSP) from abalone ( Haliotis discus hannai ). We found out that Hdh-cSP was widely expressed in multiple tissues of abalone, with highest level in the immune-like organ, hepatopancreas. V. parahemolyticus infection induced significantly elevated expression of Hdh-cSP in addition to better-characterized innate immune component genes including Rel/NF-κB, allograft inflammatory factor (ALInFa), macrophage expressed protein (MEP) and caspase-8. Importantly, the silencing of Hdh-cSP reduced the expression of these genes, suggesting that Hdh-cSP was an upstream regulatory factor in V. parahemolyticus infection. Further analysis showed that apoptosis of hemocytes was inhibited when the transcription of Hdh-cSP was knocked down, suggesting that Hdh-cSP participated in cell apoptosis by regulation of caspase 8 expression in V. parahemolyticus infection. Therefore, our study established an important role of cSP in the innate immunity against V. parahemolyticus infection in abalone. [ABSTRACT FROM AUTHOR]

Superoxide dismutases (SODs), a by-product of antioxidative defence system, protects organisms for eliminating excess reactive oxygen species (ROS) and maintaining the redox balance of immune system. The complete open reading frames (ORFs) of Cu/Zn-SOD and Mn-SOD were identified from Mytilus coruscus (designated as McSOD and MnSOD) by homologous cloning. The sequence lengths were 474bp and 687bp, encoding 157 and 228 amino acids respectively. The deduced amino acid sequences of McSOD and MnSOD shared high identities with Cu/Zn-SOD and Mn-SOD from other mollusca. The distributions of McSOD and MnSOD were detected in six tissues including adductor, hemocyte, gill, gonad, mantle and hepatopancreas, and the highest expressions were both in gills. The temporal expression of McSOD and MnSOD were up-regulated in gills under a variety of stress factors, including Vibrio parahemolyticus , Aeromonas hydrophila , Cu 2+ and Pb 2+ . After being challenged with V. Parahemolyticus , the expressions of McSOD and MnSOD were increased rapidly at the initial hours, reaching the peaks of 4.9-fold and 15.3-fold respectively, and got to the highest levels of 43.5-fold and 7.1-fold after being challenged with A. hydrophila . The highest point of McSOD mRNA appeared at 15 d after being exposed to copper (7-fold at 0.5 mg/L and 13.2-fold at 1.5 mg/L), except for 0.1 mg/L group of Cu 2+ maintaining to the normal level, but plumbum at 1 d (2.4-fold at 1.0 mg/L and 4.4-fold at 3.0 mg/L) and at 15 d (2.1-fold at 0.2 mg/L). The temporal expression peaks of MnSOD appeared differently after exposing to copper of various concentrations (0.1 mg/L at 10 d with 4.7-fold, 0.5 mg/L at 1 d with 17.9-fold and 1.5 mg/L at 3 d with 13.2-fold). Whereas in plumbum exposing treatments, the 3.0 mg/L group jumped to the peak at 1 d (18.2-fold), the 0.2 mg/L and 1.0 mg/L groups had little change and maintained at the normal level throughout the experiment. The results provided several new evidences for further understanding of the regulatory mechanism of SOD on the innate immune system in bivalve. [ABSTRACT FROM AUTHOR]

In the Viable but Non-Culturable (VBNC) state, microorganisms may survive under severe external environment. In this study, the specificity and sensitivity of PMA-LAMP assay on the detection of Vibrio Parahemolyticus ( V. parahemolyticus ) has been developed and evaluated, with further application on a number of food-borne V. parahemolyticus strains. Six primers were designed for recognizing 8 distinct targeting on tlh , tdh and trh gene. Through specific penetration through the damaged cell membrane of dead cells and intercalating into DNA, PMA could prevent DNA amplification of dead bacteria from LAMP, which enabled the differentiation of bacteria between VBNC state and dead state. The established PMA-LAMP showed significant advantage in rapidity, sensitivity and specificity, compared with regular PCR assay. The applicability had also been verified, demonstrating the PMA-LAMP was capable of detection on V. parahaemolyticus. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that participate in diverse biological processes via regulating expressions of target genes at post-transcriptional level. Amphioxus, as modern survivor of an ancient chordate lineage, is a model organism for comparative genomics study. However, miRNAs involved in regulating immune responses in Branchiostoma belcheri are largely unclear. Here, we systematically investigated the microRNAs (miRNAs) involved in regulating immune responses in the cephalochordate amphioxus ( Branchiostoma belcheri ) through next-generation deep sequencing of amphioxus samples infected with Vibrio parahemolyticus . We identified 198 novel amphioxus miRNAs, consisting of 12 conserved miRNAs, 33 candidate star miRNAs and 153 potential amphioxus-specific-miRNAs. Using microarray profiling, 14 miRNAs were differentially expressed post infection, suggesting they are immune-related miRNAs. Eight miRNAs ( bbe-miR-92a-3p , bbe-miR-92c-3p , bbe-miR-210-5p, bbe-miR-22-3p , bbe-miR-1∼bbe-miR-133 and bbe-miR-217∼bbe-miR-216 clusters) were significantly increased at 12 h post-infection, while bbe-miR-2072-5p was downregulated at 6 h and 12 h. Three miRNAs, bbe-miR-1-3p , bbe-miR-22-3p and bbe-miR-92a-3p , were confirmed to be involved in immune responses to infection by qRT-PCR. Our findings further clarify important regulatory roles of miRNAs in the innate immune response to bacterial infection in amphioxus. [ABSTRACT FROM AUTHOR]

Vibrio alginolyticus, Vibrio parahemolyticus and Photobacterium damselae subsp damselae were isolated during recurrent episodes of mass mortalities among different stages of Gilthead sea bream (Sparus aurata) and European seabass (Dicentrarchus labrax). The pathogens were recovered from the external/internal lesions of a total of 320 seeds, juvenile and adult fishes from the period of February 2013 through August 2013. Two hundred and sixty four bacterial isolates were retrieved and presumptively identified using morpho-chemical characterization and API®20NE. However, definitive molecular confirmation of V. alginolyticus was obtained through implementing collagenase gene based regular PCR technique. The total prevalence of V. alginolyticus, V. parahemolyticus and Photobacterium damselae subsp damselae among naturally infected Gilthead seabream and European seabass was 82.19%, 87.28% 10.27%, 6.79% and 7.54%, 5.93% respectively. Antibiogram has revealed that isolates were sensitive to ciprofloxacin, chloramphenicol, enrofloxacin, nalidixic acid and oxolinic acid while resistant to ampicillin, amoxicillin, and lincomycin.

Drosophila Toll and mammalian Toll-like receptors (TLRs) are a family of evolutionarily conserved immune receptors that play a crucial role in the first-line defense against intruded pathogens. Activating transcription factor 4 (ATF4), a member of the ATF/CREB transcription factor family, is an important factor that participates in TLR signaling and other physiological processes. However, in crustaceans, whether ATF4 homologs were involved in TLR signaling remains unclear. In the current study, we identified a Toll homolog PcToll2 and a novel ATF4 homolog Pc ATF4 from Procambarus clarkii , and analyzed the likely regulatory activity of Pc ATF4 in Pc Toll2 signaling. The complete cDNA sequence of PcToll2 was 4175 bp long containing an open reading frame of 2820 bp encoding a 939-amino acid protein, and the cDNA sequence of PcATF4 was 2027 bp long with an open reading frame of 1296 bp encoding a 431-amino acid protein. Pc Toll2 and human TLR4 shared the high identity and they were grouped into a cluster. Furthermore, Pc Toll2 had a close relationship with other shrimp TLRs that possessed potential antibacterial activity. PcToll2 was highly expressed in the hemocytes, heart and gills, while PcATF4 mainly distributed in gills. Upon challenge with Vibrio parahemolyticus , PcToll2 and PcATF4 together with the antimicrobial peptides of ALF1 and ALF2 were significantly up-regulated in the hemocytes, and the Pc ATF4 was translocated into the nucleus. After PcToll2 silencing and challenge with Vibrio , the translocation of Pc ATF4 into the nucleus was inhibited and the expression of ALF1 and ALF2 was reduced, but the expression of PcDorsal and PcSTAT was not affected. Furthermore, after PcATF4 knockdown and challenge with or without Vibrio , the expression of ALF1 and ALF2 was also decreased while the expression of Pc Toll2 was upregulated. These results suggested that PcToll2 might regulate the expression of ALF1 and ALF2 by promoting the import of Pc ATF4, instead of the routine transcription factor Pc Dorsal, into the nucleus participating in the immune defense against Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

The structure of a polysaccharide from Vibrio parahaemolyticus strain AN-16000 has been investigated. The sugar and absolute configuration analysis revealed d -Glc, d -GalN, d -QuiN and l -FucN as major components. The PS was subjected to dephosphorylation with aqueous 40% HF to obtain an oligosaccharide that was analyzed by 1 H and 13 C NMR spectroscopy. The HR-MS spectrum of the oligosaccharide revealed a pentasaccharide composed of two Glc residues, one QuiNAc and one GalNAc, one FucNAc, as well as a glycerol moiety. The structure of the PS was determined using 1 H, 13 C, 15 N and 31 P NMR spectroscopy; inter-residue correlations were identified by 1 H, 13 C-heteronuclear multiple-bond correlation, 1 H, 1 H-NOESY and 1 H, 31 P-hetero-TOCSY experiments. The PS backbone has the following teichoic acid-like structure: →3)- d -Gro-(1- P -6)-β- d -Glc p -(1→4)-α- l -Fuc p NAc-(1→3)-β- d -Qui p NAc-(1→ with a side-chain consisting of α- d -Glc p -(1→6)-α- d -GalpNAc-(1→ linked to the O3 position of the FucNAc residue. [ABSTRACT FROM AUTHOR]

Microbial growth variability plays an important role on food safety risk assessment. In this study, the growth kinetic characteristics corresponding to maximum specific growth rate (µmax) of 50 V. parahaemolyticus isolates from different sources and genotypes were evaluated at different temperatures (10, 20, 30, and 37°C) and salinity (0.5, 3, 5, 7, and 9%) using the automated turbidimetric system Bioscreen C. The results demonstrated that strain growth variability increased as the growth conditions became more stressful both in terms of temperature and salinity. The coefficient of variation (CV) of µmax for temperature was larger than that for salinity, indicating that the impact of temperature on strain growth variability was greater than that of salinity. The strains isolated from freshwater aquatic products had more conspicuous growth variations than those from seawater. Moreover, the strains with tlh+/tdh+/trh- exhibited higher growth variability than tlh+/tdh-/trh- or tlh+/tdh-/trh+, revealing that gene heterogeneity might have possible relations with the growth variability. This research illustrates that the growth environments, strain sources as well as genotypes have impacts on strain growth variability of V. parahaemolyticus, which can be helpful for incorporating strain variability in predictive microbiology and microbial risk assessment. [ABSTRACT FROM AUTHOR]

Innate immunity in shrimp is important in resisting bacterial infection. The NF-κB pathway is pivotal in such an immune response. This study cloned and functionally characterized the solute carrier family ( SLC ) 15 member A 4 ( LvSLC15A4 ) gene in Litopenaeus vannamei . The open reading frame of LvSLC15A4 is 1, 902 bp long and encodes a putative 633-amino acid protein, which is localized in the plasma membrane and intracellular vesicular compartments. Results of the reporter gene assay showed that LvSLC15A4 upregulated NF-κB target genes, including the immediate-early gene 1 of white spot syndrome virus, as well as several antimicrobial peptide genes , such as pen4 , CecA , AttA , and Mtk in S2 cells. Moreover, knocked-down expression of LvSLC15A4 reduced pen4 expression in L. vannamei . LvSLC15A4 down-regulation also increased the cumulative mortality of Vibrio parahemolyticus -infected L. vannamei . Furthermore, LvSLC15A4 expression was induced by unfolded protein response (UPR) in L. vannamei hematocytes. These results suggest that LvSLC15A4 participates in L. vannamei innate immunity via the NF-κB pathway and thus may be related to UPR. [ABSTRACT FROM AUTHOR]

The Cladosporium fungi, one of the largest genera of dematiaceous hyphomycetes, could produce various bioactive secondary metabolites. From the AcOEt-soluble extract of Cladosporium oxysporum 170103, three new secopatulolides (1-3) and thirteen known compounds (4-16) were obtained. Their structures were established by detailed analysis of the NMR and HR-ESI-MS data. All sixteen compounds were tested for antibacterial activity against Vibrio parahemolyticus, ergosterol (10) presented moderate effect with the minimum inhibitory concentration (MIC) of 32 μM. It can destruct the membrane integrity of Vibrio parahemolyticus to change the cell shape., (© 2022 Wiley-VHCA AG, Zurich, Switzerland.)

Vibrio parahaemolyticus , one of the major causes of vibriosis in aquatic animals, causes inflammation that may be related to tissue damage. Flagellin is the major structural component of the flagellum, and also an important virulence factor of bacteria. The filament of V. parahaemolyticus flagellum is composed of multiple flagellin subunits, including FlaA, FlaB, FlaC, FlaD, FlaE, and FlaF which are related to virulence. We produced six V. parahaemolyticus recombinant flagellin through prokaryotic expression including r V.p FlaA, r V.p FlaB, r V.p FlaC, r V.p FlaD, r V.p FlaE, and r V.p FlaF. The primary spleen and head kidney cells (including leukocytes) of Tetraodon nigroviridis were obtained, and the recombinant flagellins were used as stimulants. In this study, the recombinant V. parahaemolyticus flagellin on pro-inflammatory mediators including the release of nitric oxide (NO) and reactive oxygen species, as well as the expression of inflammatory cytokines IL-1β and IFNγ2 etc were detected. The results showed that the NO production and respiratory burst response were significantly induced after stimulation with r V.p FlaF in T. nigroviridis head kidney and spleen cells. Our results also showed that the mRNA levels of T. nigroviridis toll-like receptor 5 M (Tn TLR5M), Tn TLR5S, and downstream genes, such as myeloid differentiation factor 88 (MyD88), TNF receptor-associated factor 6 (TRAF6), interleukin-1β (IL-1β), and IFNγ2 were all significantly enhanced after stimulation with r V.p FlaF in T. nigroviridis primary cells. In addition, the NO production and alkaline phosphatase (ALP) activity were significantly increased in the liver of T. nigroviridis after the r V.p FlaF challenge in vivo. Meanwhile, in Tn TLR5M-overexpressed HEK 293 T and Cos-7 cells, the luciferase activity of the nuclear factor-kappa B (NF-κB) reporter gene was significantly enhanced after r V.p FlaF stimulation, in a time- and dose-dependent manner. However, the other five flagellin components have no inflammation-inducing ability or the effect is relatively weak. Overall, our results suggested that r V.p FlaF activates the pro-inflammatory mediators including the release of NO and reactive oxygen species, as well as the expression of inflammatory cytokines IL-1β and IFNγ2, through the Tn TLR5M-NF-κB signaling pathway in T. nigroviridis. In conclusion, this study indicated that the r V.p FlaF has a significant role in inducing the inflammation of T. nigroviridis. • Vibrio parahaemolyticus flagellin induces the inflammatory response of Tetraodon nigroviridis • V.p FlaF has the ability to activate the T. nigroviridis immune response • V.p FlaF induces the inflammatory response through the TLR5M-MyD88-NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract: Repertoires of proteins and small peptides play numerous physiological roles as hormones, antimicrobial peptides, and cellular signaling factors. The beta-thymosins are a group of small acidic peptides involved in processes such as actin sequestration, neuronal development, wound healing, tissue repair, and angiogenesis. Recent characterization of the beta thymosins as immunological regulators in invertebrates led to our identification and characterization of a beta-thymosin homologue (Tβ) from Haliotis discus discus. The cDNA possessed an ORF of 132bp encoding a protein of 44 amino acids with a molecular mass of 4977Da. The amino acid sequence shows high identity with another molluskan beta-thymosin and has a characteristic actin binding motif (LKKTET) and glutamyl donors. Phylogenetic analysis showed a close relationship with molluskan homologues, as well as its distinct identity and common ancestral origin. Genomic analysis revealed a 3 exon–2 intron structure similar to the other homologues. In silico promoter analysis also revealed significant transcription factor binding sites, providing evidence for the expression of this gene under different cellular conditions, including stress or pathogenic attack. Tissue distribution profiling revealed a ubiquitous presence in all the examined tissues, but with the highest expression in mantle and hemocyte. Immune challenge with lipopolysaccharide, poly I:C and Vibrio parahemolyticus induced beta-thymosin expression in gill and hemocytes, affirming an immune-related role in invertebrates. [Copyright &y& Elsevier]

Prevalence of faecal indicator bacteria, Escherichia coli and pathogenic bacteria, Vibrio cholerae, Vibrio parahaemolyticus and Salmonella were analysed in Vembanadu lake (98350N 768250E), along south west coast of India for a period of one year from ten stations on the southern and northern sides of a salt water regulator constructed in Vembanadu Lake in order to prevent incursion of seawater during certain periods of the year. While the northern side of the lake has a connection to the sea, the southern side is enclosed when the salt water regulator is closed. The results revealed the water body is polluted with high faecal coliform bacteria with mean MPN value ranging from 1718-7706/100 ml. E. coli, V. cholerae, V. parahaemolyticus and Salmonella serotypes such as S. paratyphi A, B, C and S. newport were isolated and this is the first report on the isolation of these Salmonella serovars from this lake. E. coli showed highest percentage of incidence (85.6-86.7%) followed by Salmonella (42-57%), V. choleare (40-45%) and V. parahaemolyticus (31.5-32%). The increased prevalence of indicator and pathogenic bacteria in the enclosed southern part of Vembanadu Lake may be resulting from the altered flow patterns due to the salt water regulator. [ABSTRACT FROM AUTHOR]