Parahaemolyticus Infection Research Articles

A case-control analysis of traceback investigations for Vibrio parahaemolyticus infections (vibriosis) and pre-harvest environmental conditions in Washington State, 2013–2018

BackgroundVibrio parahaemolyticus is a major cause of seafood-borne illness. It is naturally prevalent in brackish waters and accumulates in shellfish. Vibriosis cases are rising globally, likely due to rising temperatures. ObjectivesTo identify associations between vibriosis in Washington State and pre-harvest environmental and V. parahaemolyticus genetic measurements sampled from shellfish. MethodsSuccessful vibriosis traceback investigations were spatiotemporally matched to routine intertidal oyster (Crassostrea gigas) sampling events, which included measurements of temperature, salinity, and V. parahaemolyticus genetic targets (thermolabile hemolysin: tlh; thermostable direct hemolysin: tdh; thermostable direct-related hemolysin: trh). Unmatched sampling events were treated as controls. Associations were evaluated using logistic regression models. ResultsSystematic differences were observed across Washington harvesting zones. These included positive associations between the odds of vibriosis and all three genetic targets in South Puget Sound, with a large odds ratio (OR) = 13.0 (95% CI: 1.5, 115.0) for a 1-log10 increase in tdh when total bacterium abundance was low (tlh < 1 log10 MPN/g). A positive association also occurred for a 1 °C increase in tissue temperature OR = 1.20 (95% CI: 1.10, 1.30) while a negative association occurred for a similar increase in water temperature OR = 0.70 (95% CI: 0.59, 0.81). In contrast, the coastal bays displayed positive associations for water temperature OR = 2.16 (95% CI, 1.15, 4.05), and for a 1-log10 increase in the tdh:trh ratio OR = 5.85 (95% CI, 1.06, 32.26). DiscussionThe zonal variation in associations indicates unique pathogenic strain prominence, suggesting tdh+/trh+ strains in South Puget Sound, such as the O4:K12 serotype, and tdh+/trh− strains in the coastal bays. The temperature discrepancy between water and oyster tissue suggests that South Puget Sound pathogenic strains flourish with exposure to relatively warm air during low tide. These findings identify new ecological risk factors for vibriosis in Washington State that can be used in future prevention efforts.

Effects of Vibrio parahaemolyticus infection on physiological response, histopathology and transcriptome changes in the mud crab (Scylla paramamosain)

Mud crab (Scylla paramamosain) is an important economic species in China. Vibrio parahaemolyticus infection have caused a great economic loss in mud crab farming. The mechanism involved in the immune responses of mud crab to V. parahaemolyticus is unclear. In this study, the physiological and immune response to V. parahaemolyticus infection were investigated in S. paramamosain. The results showed that V. parahaemolyticus infection decreased total hemocyte counts, led to cytological damage, and caused high mortality. Transcriptome analysis showed that 1327 differentially expressed genes (DEGs), including 809 up-regulated and 518 down-regulated ones, were obtained after V. parahaemolyticus challenge. These DEGs were mainly involved in the immune response and infectious disease. Additionally, transcriptome analysis revealed that Toll, immune deficiency (IMD), and prophenoloxidase signalling pathways played essential roles in antibacterial immunity against V. parahaemolyticus infection in mud crab.

A double chitin catalytic domain-containing chitinase targeted by c-Jun is involved in immune responses in shrimp

Chitinases are a group of chitin-degrading enzymes widely distributed in organisms. Chitinases containing two chitin catalytic domains have been widely found in arthropods but their functions remain unclear. In this study, a member of these chitinases from Litopenaeus vannamei (dChi) was identified and functionally studied in the context of immunity. The promoter of dChi contained activator protein 1 (AP-1) binding sites and could be regulated by c-Jun. The recombinant dChi protein showed no bacteriostatic activity in vitro but knockdown of dChi in vivo increased the mortality of shrimp and the bacterial load in tissues after Vibrio parahaemolyticus infection, suggesting that dChi could play a positive role in antibacterial responses. However, silencing of dChi expression significantly decreased the mortality of WSSV-infected shrimp and down-regulated the viral load in tissues, indicating that dChi could facilitate WSSV infection. We further demonstrated that dChi was involved in regulation of the bacterial phagocytosis of hemocytes and expression of a series of immune related transcription factors and antimicrobial peptides. These indicated that the roles of dChi in antibacterial responses and anti-WSSV responses in vivo could result from its regulatory effects on the immune system. Taken together, the current study suggests that double chitin catalytic domain-containing chitinases could be important players in immune regulation in crustaceans.

Molecular and Functional Diversity of Crustin-Like Genes in the Shrimp Litopenaeus vannamei.

Crustins are crustacean cationic cysteine-rich antimicrobial peptides that contain one or two whey acidic protein (WAP) domain(s) at the carboxyl terminus and mainly show antimicrobial and/or proteinase inhibitory activities. Here, we performed genome and transcriptome screening and identified 34 full-length crustin-like encoding genes in Litopenaeus vannamei. Multiple sequence analysis of the deduced mature peptides revealed that these putative crustins included 10 type Ia, two type Ib, one type Ic, 11 type IIa, three type IIb, four type III, one type IV, one type VI, and one type VII. These putative crustins were clustered into different groups. Phylogenetic analysis, considering their domain composition, showed that different types of crustin-like genes in crustaceans might be originated from the WAP core region, along with sequence insertion, duplication, deletion, and amino acid substitution. Tissue distribution analysis suggested that most crustin-like genes were mainly detected in immune-related tissues while several crustin-like genes exhibited tissue-specific expression patterns. Quantitative PCR analysis on 15 selected crustin-like genes showed that most of them were apparently upregulated after Vibrio parahaemolyticus or white spot syndrome virus (WSSV) infection. One type Ib crustin-like gene, mainly expressed in the ovary, showed the highest expression levels before the gastrula stage and was hardly detected after the limb bud stage, suggesting that it was a maternal immune effector. Collectively, the present data revealed the molecular and functional diversity of crustins and their potential evolutionary routes in crustaceans.

Taiman negatively regulates the expression of antimicrobial peptides by promoting the transcription of cactus in Macrobrachium nipponense

In insects, Taiman (Tai) participates in the juvenile hormone, 20-hydroxyecdysone, insulin, and Hippo signaling pathways. However, the role of Tai in crustacean innate immunity is less known. In this study, four Tai isoforms (MnTai-A, MnTai-B, MnTai-C, and MnTai-D) produced by alternative splicing were identified from Macrobrachium nipponense. The obtained genome sequences indicated that MnTai DNA has more than 20 exons and 19 introns. The second to last (-exon2) and the third to last (-exon3) exons can be alternatively spliced. The loss of -exon2 or -exon3 produces MnTai-B or MnTai-C, respectively. Both exons are absent in MnTai-D. The full-length cDNA of MnTai-A (including all exons) was 6894 bp with an open reading frame of 4998 bp that encoded a protein of 1665 amino acids. MnTaiA contains the conservative structure of the Tai family and clustered with nuclear receptor coactivator from shrimp. All these four isoforms were widely distributed in a variety of tissues with the highest expression level in the hepatopancreas except MnTaiC. The transcriptional levels of total Tai genes (designated as MnTaiT) in the hepatopancreas and gills were regulated by bacterial or viral challenge. Knockdown of MnTaiT increased the expression of anti-microbial peptides (AMPs) during Vibrio parahaemolyticus infection. Further study indicated that the negative regulation of AMP gene expression by prawn Tai was mediated through its positive regulation of cactus. Our research provides valuable information that prawn Tai isoforms are involved in innate immunity.

Rapid identification and detection of Vibrio parahaemolyticus via different types of modus operandi with LAMP method in vivo

PurposeVibrio parahaemolyticus, an easy-ignored food-borne pathogen, can cause bacterial outbreaks and human disease during early-stage infection. In this study, we aimed to evaluate the detection efficiency of loop-mediated isothermal amplification (LAMP) as an emerging technique to directly detect V. parahaemolyticus infection in mammalian hosts and assess its potential in clinical applications.MethodsA LAMP assay was used for rapid identification of V. parahaemolyticus in a variety of mouse models in which animals were infected via the digestive tract, wounds, or through general infection, and the results were compared with routine analytical methods.ResultsOur results confirmed that the LAMP assay was capable of detecting V. parahaemolyticus in different mouse organs independent of the source of bacteria, although its sensitivity depended on the route of infection and the organ affected. Foodborne-derived V. parahaemolyticus was the most sensitive route, with the small intestine being the most sensitive organ. The LAMP assay indicated that V. parahaemolyticus that spread through the blood stream had the most serious consequences during early-stage infection. Positive LAMP results were identified in all blood samples from i.v. injected mice. Furthermore, the LAMP method could directly detect trace quantities of V. parahaemolyticus in fresh peripheral blood while conventional methods failed to do so, thereby shortening the time-to-result from days to minutes.ConclusionsIn this study, we demonstrated that the LAMP assay was effective in speeding up the detection of V. parahaemolyticus. Instead of being a secondary method to assist in the clinic, the LAMP assay has potential for use as the primary technique for rapid detection of V. parahaemolyticus in the future.

Enhancement of shrimp health and immunity with diets supplemented with combined probiotics: application to Vibrio parahaemolyticus infections.

The application of probiotics for disease control in aquaculture is now a convincing approach towards replacement of antibiotics, which can cause adverse effects in aquatic animals and humans. In this study, we combined 2 probiotics, Lactobacillus acidophilus and Saccharomyces cerevisiae, with shrimp feed to create 2 formulas (WU8 and WU9), which were fed for 10 d to juvenile shrimp Penaeus vannamei. The shrimps were then subjected to a challenge infection with Vibrio parahaemolyticus, the causative agent of acute hepatopancreas necrosis disease (AHPND). The protective effects of probiotics against bacterial infection were investigated through histopathology of the hepatopancrease and immunological evaluation of shrimp. Both WU8 and WU9 probiotic mixtures (1:1, at 108 and 109 CFU kg diet-1) increased blasenzellen hepatopancreatic epithelial cells and reduced pathology caused by AHPND. After 10 d of feeding, hemocyte parameters, including the total hemocyte count, percent of granular hemocytes, and phenoloxidase activity, increased significantly and were still increasing at 24 h post infection. Crustin and penaeidin 3 genes were also highly upregulated in hemocytes before and after 24 h of bacterial challenge and significantly upregulated in the hepatopancreas 1 to 5 d post-infection. A significantly higher survival rate was observed in shrimp fed with the probiotic supplemented diet (>90%) in comparison to the control group (60%). In conclusion, probiotic mixtures of L. acidophilus and S. cerevisiae reduced hepatopancreas pathology and protected shrimp from a challenge with AHPND.

The sterol regulatory element binding protein homolog of Penaeus vannamei modulates fatty acid metabolism and immune response

The sterol regulatory element binding proteins (SREBPs) transcription factors family, which regulate the expression of genes involved in cellular lipid metabolism and homeostasis, have recently been implicated in various physiological and pathophysiological processes such as immune regulation and inflammation in vertebrates. Consistent with other invertebrates, we identified a single SREBP ortholog in Penaeus vannamei (designated PvSREBP) with transcripts ubiquitously expressed in tissues and induced by lipopolysaccharide (LPS), Vibrio parahaemolyticus and Streptococcus iniae. In vivo RNA interference (RNAi) of PvSREBP attenuated the expression of several fatty acid metabolism-related genes (i.e., cyclooxygenase (PvCOX), lipoxygenase (PvLOX), fatty acid binding protein (PvFABP) and fatty acid synthase (PvFASN)), which consequently decreased the levels of total polyunsaturated fatty acids (ΣPUFAs). In addition, PvSREBP silencing decreased transcript levels of several immune-related genes such as hemocyanin (PvHMC) and trypsin (PvTrypsin), as well as genes encoding for heat-shock proteins (i.e., PvHSP60, PvHSP70 and PvHSP90). Moreover, in silico analysis revealed the presence of SREBP binding motifs on the promoters of most of the dysregulated genes, while shrimp depleted of PvSREBP were more susceptible to V. parahaemolyticus infection. Collectively, we demonstrated the involvement of shrimp SREBP in fatty acids metabolism and immune response, and propose that PvSREBP and PvHMC modulate each other through a feedback mechanism to establish homeostasis. The current study is the first to show the dual role of SREBP in fatty acid metabolism and immune response in invertebrates and crustaceans.

Autophagy Dually Induced by AMP Surplus and Oxidative Stress Enhances Hemocyte Survival and Bactericidal Capacity via AMPK Pathway in Crassostrea hongkongensis.

Crassostrea hongkongensis (Hong Kong oyster) is an ecologically and economically valuable shellfish endemic to South/Southeast Asia. Due to ocean acidification and warming waters, they have become increasingly vulnerable to invading microbes including Vibrio parahaemolyticus, a significant foodborne human pathogen. In recent years, outbreaks of V. parahaemolyticus have emerged as a perennial phenomenon in parts of the world, necessitating to better understand the biology of host-pathogen interactions in this under-examined marine invertebrate. Although an immunologically relevant autophagy apparatus has been identified in Crassostrea gigas, an evolutionarily close mollusk cousin, the precise mechanistic details of C. hongkongensis autophagy during V. parahaemolyticus infection are still wanting. Here, we compellingly demonstrated that in vivo V. parahaemolyticus challenge robustly triggered autophagic signaling in C. hongkongensis hemocytes peaking at 6 h post-infection, which subsequently promoted bacterial clearance and dampened premature apoptosis. Simultaneously, a large surplus of adenosine monophosphate (AMP) and elevations in reactive oxygen species (ROS, specifically mitochondrial O2– and cellular H2O2) formation were observed post-infection. Extrinsically applied AMP and ROS could synergistically induce AMP-activated protein kinase (AMPK) phosphorylation to stimulate downstream autophagic events. V. parahaemolyticus infection-induced autophagy was pharmacologically shown to be AMPK-dependent in vivo. Overall, our results establish autophagy as a crucial arm of host defense against Vibrio infections in mollusks, and provide new insights into the underappreciated roles of ROS and AMP as co-regulators of autophagy.

SpATF2 participates in maintaining the homeostasis of hemolymph microbiota by regulating dual oxidase expression in mud crab

Activating transcription factors 2 (ATF2) is a transcription factor of the members of ATF/CREB family that is phosphorylated and activated by the mitogen-activated protein kinase (MAPK) in responding to the stimulation of stimuli. In present study, SpATF2 from mud crab (Scylla paramamosain) was identified and studied. The open reading frame of SpATF2 with 2136 bp in length encodes a protein with 711 amino acids. The SpATF2 protein includes the putative zinc finger domain in the N-terminus and bZIP type DNA-binding domain in the C-terminal. Tissue distribution of SpATF2 transcripts showed that SpATF2 was ubiquitously expressed in all examined tissues of the untreated mud crabs, with the highest expression levels in muscle and hepatopancreas. The transcriptional level of SpATF2 was up-regulated in the hemocytes after Vibrio parahemolyticus or WSSV infection. Reporter gene assays indicated that SpATF2 could activate the expression of dual oxidase (SpDuox1) in S. paramamosain. The RNA interference (RNAi) of SpATF2 significantly decreased the expression of SpDuox1, and consequently reduced reactive oxygen species production thereby significantly increased the bacterial load in the hemolymph of mud crabs. Similarly, significant reduction in bacterial clearance of hemolymph was observed after the V. parahemolyticus infection in SpATF2 knockdown mud crabs. This study showed that SpATF2 played a vital role in maintaining homeostasis of the hemolymph microbiota through regulating the expression of dual oxidase of mud crab.

The immune function of a novel crustin with an atypical WAP domain in regulating intestinal microbiota homeostasis in Litopenaeus vannamei

Crustins are a family of antimicrobial peptides (AMP) with multiple functions, including antimicrobial activity, capability of protease inhibition, phagocytosis promotion, and wound healing in crustaceans. Till present, several members of crustins have been identified and their activities were studied. However, there are still less investigations on how they play functions in vivo. Here, we identified a novel crustin with an atypical WAP domain, LvCrustin Ⅰ-1, which is mainly distributed in tissues, including intestine, gill, epidermis and stomach of the shrimp Litopenaeus vannamei. The expression level of LvCrustin Ⅰ-1 was significantly up-regulated at 3 h, 6 h, 12 h, and 24 h after Vibrio parahaemolyticus infection. Knockdown of LvCrustin Ⅰ-1 with dsRNA resulted in a significant increase of the bacteria number in hepatopancreas of shrimp upon V. parahaemolyticus infection, showing that LvCrustin Ⅰ-1 participated in pathogen infection process. Recombinant LvCrustin Ⅰ-1 protein showed microorganism-binding activity rather than antibacterial activity against tested bacteria. Furthermore, significant difference existed between the intestinal microbiota in shrimp before and after LvCrustin Ⅰ-1 knockdown based on the result of alpha and NMDS analyses. Knockdown of LvCrustin Ⅰ-1 increased the proportion of Demequina, Nautella, Propionibacterium, Anaerospora and decreased the proportion of Bacteroidia and Vibrio. These data suggest that LvCrustin Ⅰ-1 might perform its immunological function through modulation of the intestinal microbiota homeostasis rather than direct inhibition of bacterial growth in shrimp.

Transcriptome and metabolome integration analysis of mud crab Scylla paramamosain challenged to Vibrio parahaemolyticus infection

Vibrio parahaemolyticus (V. parahaemolyticus) is a common pathogen for marine crustacean, which causes severe illnesses in aquatic animals. Therefore, it is meaningful to explore the mechanism during V. parahaemolyticus infection. In this study, to investigate the immune responses of mud crab Scylla paramamosain (S. paramamosain) to V. parahaemolyticus, we established the metabolic and transcriptional profiles of mud crab hemocytes challenged with V. parahaemolyticus. The results indicated that V. parahaemolyticus infection could induce a series of metabolism alterations at both metabolome and transcriptome levels, including biosynthesis of amino acids and Aminoacyl-tRNA, Purine and pyrimidine metabolism, TCA cycle and glutamine metabolism. In this context, through the integration of metabolomics and transcriptomics, our study provided a more comprehensive understanding of the biological process in mud crab against pathogen infection.

Molecular cloning, mRNA expression and functional characterization of a catalase from Chinese black sleeper (Bostrychus sinensis)

In this study, the catalase gene of Chinese black sleeper Bostrychus sinensis (termed as BsCat) was sequenced and characterized. The BsCat, which encodes 525 amino acids, contains a catalase proximal active site signature domain (64FDRERIPERVVHAKGAG80) and a catalase proximal heme-ligand signature domain (354RLFAYPDTH362). The BsCat exhibits high sequence similarity with Cat of other species. Phylogenetic tree reconstruction revealed a close evolutionary relationship of BsCat to catalase genes of other fishes. The results of Real-time PCR showed that the BsCat gene was constitutively expressed in most organs of B. sinensis, with predominant expression detected in liver, followed by peripheral blood and spleen. Moreover, the BsCat gene was significantly changed after either poly (I:C) stimulation or Vibrio parahemolyticus infection in peripheral blood, head kidney, liver and spleen. The enzymatic activity of purified recombinant BsCat (rBsCat) was 2261 ± 96 U/mg. The rBsCat exhibits optimum enzymatic activity at 15 °C and pH 7.0. Our results suggested that the BsCat is involved in the antioxidant defense and host immune response of Chinese black sleeper during pathogen invasion.

Identification and functional study of an LRR domain containing membrane protein in Litopenaeus vannamei

Leucine-rich repeat (LRR) is a vital structure in some pattern recognition receptors such as TLRs, NLRs and newly reported LRR-containing proteins. Apart from some limited reported LRR-containing proteins, most of LRR proteins, especially immune-related proteins, remain uncharacterized functionally. In the present study, a transmembrane protein containing several LRR motifs, designated as LvLRRm, was identified from the shrimp Litopenaeus vannamei. LvLRRm contained a long signal peptide, one LRRNT region, 12 LRR motifs, one LRRCT region and a transmembrane region. The transcripts of LvLRRm were widely distributed in all tested tissues of shrimp and they were responsive to Vibrio parahaemolyticus infection in several immune-related tissues including Oka, intestine, gill and hemocytes. Knockdown of LvLRRm by dsRNA interference led to a decreased survival rate of shrimp infected by Vibrio parahaemolyticus and an increased in vivo Vibrio propagation. Meanwhile, knockdown of LvLRRm also down-regulated the expression levels of genes involved in antibacterial immune signaling pathways, including the transcription factors LvDorsal and LvRelish, and several antimicrobial peptides. These data suggested that LvLRRm played important roles in shrimp against Vibrio infection, which was probably functioning through activation of antibacterial immune signaling pathways. The present study provided new evidence to elucidate the immune function of LRR-containing proteins in invertebrates.

Managing the risk of Vibrio parahaemolyticus infections associated with oyster consumption: A review.

Vibrio parahaemolyticus is a Gram-negative bacterium that is naturally present in the marine environment. Oysters, which are water filter feeders, may accumulate this pathogen in their soft tissues, thus increasing the risk of V. parahaemolyticus infection among people who consume oysters. In this review, factors affecting V. parahaemolyticus accumulation in oysters, the route of the pathogen from primary production to consumption, and the potential effects of climate change were discussed. In addition, intervention strategies for reducing accumulation of V. parahaemolyticus in oysters were presented. A literature review revealed the following information relevant to the present study: (a) managing the safety of oysters (for human consumption) from primary production to consumption remains a challenge, (b) there are multiple factors that influence the concentration of V. parahaemolyticus in oysters from primary production to consumption, (c) climate change could possibly affect the safety of oysters, both directly and indirectly, placing public health at risk, (d) many intervention strategies have been developed to control and/or reduce the concentration of V. parahaemolyticus in oysters to acceptable levels, but most of them are mainly focused on the downstream steps of the oyster supply chain, and (c) although available regulation and/or guidelines governing the safety of oyster consumption are mostly available in developed countries, limited food safety information is available in developing countries. The information provided in this review may serve as an early warning for managing the future effects of climate change on the safety of oyster consumption.

Development of a monoclonal antibody for specific detection of Vibrio parahaemolyticus and analysis of its antigen

Vibrio parahaemolyticus is a major foodborne pathogen worldwide. Contamination of V. parahaemolyticus in foods must be detected as quickly as possible because raw seafood, a major source of V. parahaemolyticus infection, is shipped immediately after production due to its short expiration date. In this study, we generated monoclonal antibodies (mAbs) against V. parahaemolyticus to develop a rapid and specific detection assay. Obtained mAbs were categorized into four groups according to their specificity. Of the groups, Group 1 (mAb VP7, VP11, and VP24) reacted to O1–O12 of V. parahaemolyticus without cross-reaction with human pathogenic Vibrio spp. (V. alginolyticus, V. cholerae, V. fluvialis, V. furnissii, V. mimicus, and V. vulnificus). We developed an immunochromatographic (IC) strip for the rapid detection of V. parahaemolyticus in the field using VP7 as a membrane-immobilized antibody and VP24 as a colloidal gold-conjugated antibody. The IC strip detected any and all serogroups (O1 to O12) or isolates (clinical, food, and environmental strains) of V. parahaemolyticus, regardless of the presence of virulence factors thermostable direct hemolysin (TDH) or TDH-related hemolysin (TRH). It did not cross-react with any other non-V. parahaemolyticus strains tested. To elucidate the target of the IC strip, we analyzed the antigen recognized by these mAbs. Group 1 mAbs showed two specific bands at molecular masses of approximately 11 and 16 kDa by western blotting analysis. Nano liquid chromatography mass spectrometry (LC-MS)/MS analysis revealed that the candidate antigen recognized by these mAbs was outer membrane (OM) lipoprotein Q87G48. We verified that mAb VP7 detected His-tagged OM lipoprotein synthesized by reconstituted cell-free protein synthesis reagent. Reactivity to an N-terminus deletion form and protease digestion form of the OM lipoprotein showed that the extent of epitope recognized by VP mAbs was 22nd–41st amino acids (AAs) from N-terminus of the OM lipoprotein, with the sequence “22SDDAATANAAKLDEL36.” This region was also confirmed to be a V. parahaemolyticus-specific sequence by comparing putative orthologs of OM lipoprotein among Vibrio spp. The C-terminus deletion form (1st–39th AAs) including the sequence primarily recognized by VP mAbs (22nd–36th AAs) showed poor reactivity, indicating that the sequence after 40 residues of OM lipoprotein is also important for recognition by VP mAbs and VP mAbs recognize a conformational epitope. Bioinformatics research demonstrated that the OM lipoprotein is an ortholog of the lpp protein conserved throughout many bacteria. Lpp is an abundant and constitutively expressed protein and exists on the bacterial surface, suggesting it may be a good target for detection of V. parahaemolyticus.

Characterization of calcineurin A and B genes in the abalone, Haliotis diversicolor, and their immune response role during bacterial infection.

Calcineurin (CN) is known to be involved in many biological processes, particularly, the immune response mechanism in many invertebrates. In this study, we characterized both HcCNA and HcCNB genes in Haliotis diversicolor, documented their expression in many tissues, and discerned their function as immune responsive genes against Vibrio parahaemolyticus infection. Similar to other mollusk CNs, the HcCNA gene lacked a proline-rich domain and comprised only one isoform of its catalytic unit, in contrast to CNs found in mammals. HcCNB was highly conserved in both sequence and domain architecture. Quantitative PCR and in situ hybridization revealed that the genes were broadly expressed and were not restricted to tissues traditionally associated with immune function. Upon infection of H. diversicolor with V. parahaemolyticus (a bacteria that causes serious disease in crustaceans and mollusks), both HcCNA and HcCNB genes were highly up-regulated at the early phase of bacterial infection. HcCNB was expressed significantly higher than HcCNA in response to bacterial challenge, suggesting its independent or more rapid response to bacterial infection. Together, the two CN genes are unique in their gene structure (particular HcCNA) and distribution in mollusk species and likely function as immune responsive genes along with many other genes that are enhanced in the early phase of V. parahaemolyticus infection in abalone.

Effect in white shrimp Litopenaeus vannamei of Eleutherine bulbosa (Mill.) Urb. Powder on immune genes expression and resistance against Vibrio parahaemolyticus infection

This study investigated the influence of Eleutherine bulbosa (Mill.) Urb. on the immune responses, bacterial population in the intestines, and resistance of white shrimp, Litopenaeus vannamei, against infection with Vibrio parahaemolyticus. Shrimp were fed with three dosages of powder, at 6.25 g kg–1 (P6.25), 12.5 g kg–1 (P12.5), and 25 g kg–1 (P25). One dosage of the crude extract was provided, 1.25 g kg–1 (E1.25), and the controls without administration of E. bulbosa consisted of a positive control (PC) and a negative control (NC). Feed supplementation was carried out for 30 days; then shrimp from all treatments were challenged by intramuscular injection with V. parahaemolyticus (106 cfu/mL), except for the NC. The results showed that supplementation with the powder and extract of E. bulbosa for 30 days resulted in significantly higher (P < 0.05) immune responses (total hemocyte count (THC), phenoloxidase activity (PO), respiratory bursts (RBs)), gene expression (prophenoloxidase (proPO), lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP)), and total bacterial count (TBC) compared to PC/NC. In post challenge testing, there were significantly higher levels for THC, PO, RBs, proPO, LGBP, and PE (peroxinetin), and the treatments were able to suppress V. parahaemolyticus in the intestines, hepatopancreas, and muscles and to reduce damage to the muscles and hepatopancreas. The survival rate with P12.5 was significantly higher compared to the other treatments. It was concluded that the shrimp receiving supplementation with the powder and extract of E. bulbosa had increased immunity and resistance against V. parahaemolyticus infection, with the best dosage being the P12.5 treatment.

Genomic epidemiology of domestic and travel-associated Vibrio parahaemolyticus infections in the UK, 2008–2018

Globally, V. parahaemolyticus is the most prevalent food-poisoning bacterium associated with seafood consumption. To date, the epidemiology of Vibrio parahaemolyticus infections in the UK has remained unexplored. Here we analysed archived V. parahaemolyticus strains isolated from domestic infections and travellers into the UK from 2008 to 2018 and who had submitted clinical samples to Public Health England (PHE)'s Gastrointestinal Bacteria Reference Unit. A total of 48 strains were retrieved from the PHE strain collection, confirmatory tested by PCR and analysed by genome-wide phylogeny using a global collection of genomes with representative strains from the major epidemic clones. Most reported infections entering the UK originated from travellers returning from Southeast Asia, however cases were also reported to have potentially originated from the USA, Cuba, India, Turkey, Caribbean, France, Slovenia and also within the UK, highlighting the wide geographical spread of infections. A large genetic diversity of V. parahaemolyticus strains was observed, with sequence type 3 (ST3; pandemic group) strains the most common sequence type (23 of the 48 analysed strains, 47.9%). The majority of strains were tdh positive (25/48, 71%), with 5 isolates positive for both haemolysin genes (tdh and trh), with 2 isolates trh positive only (5.7%). Six isolates did not possess either haemolysin gene. We demonstrate that whole genome sequencing can be effective to type certain human pathogens entering a traditionally considered “non-endemic” county and this was captured via passive epidemiological surveillance systems. Such approaches may potentially provide a useful snapshot of the diversity of a given pathogen group circulating worldwide.

Two Wnt genes regulate the expression levels of antimicrobial peptides during Vibrio infection in Macrobrachium nipponense

The Wnt signal transduction pathway is involved in a wide variety of cellular processes, including cell proliferation, differentiation, apoptosis, and immunity against microbial infection. In the current study, we cloned and characterized two Wnt homologues (Mn-Wnt4 and Mn-Wnt16) in Macrobrachium nipponense. The full length cDNA of Mn-Wnt4 was 3144 bp with a 1074 bp open reading frame (ORF) that encoded a protein containing 358 amino acid residues. The full length cDNA of Mn-Wnt16 transcript was 2893 bp with a 1281 bp ORF that encoded a 427 amino acid protein. Mn-Wnt4 and Mn-Wnt16 proteins contained a highly conserved WNT1 domain. Tissue distribution analysis showed that Mn-Wnt4 and Mn-Wnt16 were highly expressed in the stomach. The transcriptional levels of Mn-Wnt4 and Mn-Wnt16 in the stomach were upregulated at most tested time points after bacterial (Staphylococcus aureus and Vibrio parahaemolyticus) and viral (White spot syndrome virus) infection. Moreover, the expression levels of some antimicrobial peptides (AMPs) (including anti-lipopolysaccharide factor [ALF] and crustin [CRU]) were upregulated after V. parahaemolyticus infection. We further used dsRNA-mediated RNA interference technology to explore the relationship between these two Wnt genes and the expression levels of AMPs during V. parahaemolyticus infection. Mn-Wnt4 knockdown could significantly inhibit the expression of ALF1 and CRU4 in the stomach of V. parahaemolyticus-injected prawns, whereas Mn-Wnt16 silencing could result in the inhibition of the expression level of CRU3 and CRU4 in the stomach of V. parahaemolyticus-infected prawns. These findings indicated that the Wnt gene family might participate in the body's innate immune response to Vibrio infection by regulating the synthesis of a variety of AMPs. Our study will help to understand the role of the Wnt signaling pathway in the immune response of crustaceans.