Vibrio Parahaemolyticus Infection Research Articles

Genome-wide identification and expression profiling of Wnt gene family in Neocaridina denticulata sinensis

Wnt proteins are a class of hydrophobic secreted glycoproteins involved in diverse important biological processes, such as tissue formation and regeneration, embryonic development and innate immunity. The Wnt gene family has an early origin and is present in all deuterostomes. In the process of evolution, the phenomenon of gene expansion, contraction and adaptive evolution occurs in the Wnt gene family. In the current study, eleven Wnt genes (NdWnt1-2, NdWnt4-7, NdWnt9-11, NdWnt16, and NdWntA) belonging to different subfamilies were obtained based on the genomic and transcriptomic data of Neocaridina denticulata sinensis. Then the expression patterns of all NdWnts were analyzed in various tissues, at different developmental stages and under different stresses. The expression profiles of NdWnts at different developmental stages showed that most NdWnt genes were initially expressed at gastrula stage, and the expression of NdWnt5 and NdWnt16 throughout all developmental stages. The spatial expression of NdWnt genes presented tissue specificity. They were mainly expressed in four tissues, namely gill, intestines, ovary and eyestalk. After Vibrio parahemolyticus infection and under copper exposure, the expression levels of five NdWnts (NdWnt1, NdWnt5, NdWnt10, NdWnt16 and NdWntA) were variable. Our findings enrich the research on the Wnt gene family of N. denticulata sinensis and provide valuable insights into relationship between structure and function of Wnt genes in crustaceans.

Evaluation of Therapeutic Efficiency of Stylicin against Vibrio parahaemolyticus Infection in Shrimp Penaeus vannamei through Comparative Proteomic Approach.

The shrimp immune system defends and protects against infection by its naturally expressing antimicrobial peptides. Stylicin is a proline-rich anionic antimicrobial peptide (AMP) that exhibits potent antimicrobial activity. In this study, stylicin gene was isolated from Penaeus vannamei, cloned into vector pET-28a ( +), and overexpressed in Escherichia coli SHuffle T7 cells. The protein was purified and tested for its antibiofilm activity against shrimp pathogen Vibrio parahaemolyticus. It was resulted that the recombinant stylicin significantly reduced the biofilm formation of V. parahaemolyticus at a minimum inhibitory concentration (MIC) of 200µg. Cell aggregation was observed by using scanning electron microscopy and confocal laser scanning microscopy, and it was resulted that stylicin administration significantly affects the cell structure and biofilm density of V. parahaemolyticus. In addition, real-time PCR confirmed the downregulation (p < 0.05) of genes responsible for growth and colonization. The efficacy of stylicin was tested by injecting it into shrimp challenged with V. parahaemolyticus and 7days after infection, stylicin-treated animals recovered and survived better in both treatments (T2-100µg stylicin, - 68.8%; T1-50µg stylicin, 60%) than in control (7%) (p < 0.01). Comparative proteomic and mass spectrometry analysis of shrimp hemolymph resulted that the expressed proteins were involved in cell cycle, signal transduction, immune pathways, and stress-related proteins representing infection and recovery, and were significantly different in the stylicin-treated groups. The result of this study suggests that the stylicin can naturally boost immunity and can be used as a choice for treating V. parahaemolyticus infections in shrimp.

A novel chitinase Chi6 with immunosuppressive activity promotes white spot syndrome virus (WSSV) infection in Penaeus vannamei

Chitinases, a group of glycosylase hydrolases that can hydrolyze chitin, are involved in immune regulation in animals. White spot syndrome virus (WSSV) causes huge losses to crustacean aquaculture every year. We identified a novel chitinase Chi6 from Pacific white shrimp Penaeus vannamei, which contains a catalytic domain but no chitin-binding domain. The Chi6 expression was regulated by multiple immune signaling pathways and increased after immune stimulations. Silencing of Chi6 by RNAi in vivo did not affect Vibrio parahaemolyticus infection, but significantly increased the survival rate of WSSV-infected shrimp. The expression of multiple WSSV immediate early and structural genes was also decreased upon Chi6 silencing. The recombinant Chi6 protein showed no effect on bacterial growth but could attenuate shrimp hemocyte phagocytosis. The mRNA levels of several key elements and downstream genes of the MAPK and Dorsal pathways in Chi6-silenced shrimp were significantly up-regulated, suggesting an inhibitory effect of Chi6 on humoral immune response. Moreover, Chi6 enhanced the regulatory effect of Dorsal on the expression of WSSV ie1 gene. Therefore, Chi6 promotes WSSV infection through immunosuppression and regulation of WSSV gene expression. Targeting Chi6 could be a potential strategy for controlling WSSV disease in shrimp farming.

3-Hydroxybutyrate helps crayfish resistant to Vibrio parahaemolyticus infection in versatile ways

The bacterial storage compound poly-β-hydroxybutyrate (PHB) is a potential bio-control agent in aquaculture. It has been reported that PHB benefit to the survival and growth, and improve their immunity of aquatic animals. However, the cellular and molecular regulation mechanisms of PHB in immunity process remain unclear. This study investigated the immune mechanism of hemocytes regulated by Halomonas-PHB (PHB-HM) and PHB monomer 3-HB. Red claw crayfish Cherax quadricarinatus was used as the experimental animal in cytological study. Fluorescence microscopy and flow cytometry (FCM) analysis indicated that PHB-HM labeled with fluorescein isothiocyanate (FITC) could be engulfed by granulocytes (Gs) and semi-granulocytes (SGs) upon in vitro incubation. Transmission electron microscopy (TEM) further showed the ongoing degradation of PHB granules inside Gs and SGs after the injection of PHB-HM into crayfish sinus, but phagocytosis of PHB-HM by hyalinocyte (H) was not observed. Therefore, Gs and SGs are considered the main effector cells of cellular immunity induced by PHB-HM, and SGs likely played a particular important role in this process. To study the biosafety and molecular mechanism of PHB monomer 3-HB, hemocyte viability and expression of the related genes were determined after being exposed to 0–1 mg/mL of 3-HB, and Vibrio parahaemolyticus (VP) was used as the pathogenic bacterium. The results confirmed that 3-HB had no toxic effect on hemocytes by means of cell viability assay, and supplementation with 1 mg/mL of 3-HB suppressed the growth rate of VP in TSB medium. Moreover, injection of 3-HB into the blood sinus of crayfish remarkably improved the phagocytic rate of Gs and SGs on VP. Furthermore, transcriptome assay was designed to illuminate the molecular mechanism of 3-HB regulation using red swamp crayfish Procambarus clarkii as experimental animals. RNA-seq analysis and qRT-PCR verification revealed that the microtubule and cytoskeleton-related genes were high expressed 3 h after 3-HB injection, indicating both genes might involve in building up the innate immunity. In summary, bacterial storage PHB could be phagocytosed by main effector blood cells and likely to be degraded within the cells. 3-HB helped the crayfish resistant to pathogens through improving phagocytosis, suppressing the growth of pathogenic bacteria, and increasing the expression of microtubule-related genes. The findings in this work provide cytological and molecular evidence which will facilitate the application of PHB and 3-HB as immune-control agents in farming of aquatic animals.

Combined Therapy of Probiotic Microcapsules and Bomidin in Vibrio parahaemolyticus-Infected Rats.

With the discovery of more and more drug-resistant bacterial strains, there is an urgent need for safer and more effective alternative treatments. In this study, antibacterial peptides and probiotic microcapsules were combined to treat gastrointestinal inflammation caused by Vibrio parahaemolyticus infection. To improve the stability of probiotics in the gastrointestinal tract, two types of mixed natural anionic polysaccharides and chitosan were used as carriers to embed the probiotics. Taking Lacticaseibacillus casei CGMCC1.8727 microcapsules with good performance as the research object, the in vitro characteristics of the microcapsules were studied via acid resistance test and intestinal release test. The microcapsules were then tested for in vivo treatment in combination with the antibacterial peptide, bomidin, and the therapeutic effects were compared among microencapsulated probiotics, free probiotics, and probiotics in combination with bomidin. Microencapsulation was successfully manufactured under suitable processing parameters, with the product particle size being 2.04 ± 0.2743 mm. Compared with free probiotics, microencapsulation significantly improved the activity and preservation stability of the probiotics under simulated gastrointestinal conditions. Microencapsulated probiotics showed better therapeutic effects than free probiotics in vivo. Microcapsules combined with antimicrobial peptides accelerated the elimination of bacteria in vivo. This study provides a reference for anti-inflammatory treatment, especially for the treatment of gastrointestinal diseases.

Molecular characterization and functional study of the NLRP3 inflammasome genes in Tetraodon nigroviridis

Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is an important inflammasome in mammals, which is of great significance to eliminate pathogens. However, the research of the NLRP3 inflammasome in teleost is limited. Tetraodon nigroviridis has the characteristics of small genome and easy feeding, which can be used as a model for the study of fish immune function. In present study, three NLRP3 inflammasome component genes (NLRP3, ASC and caspase-1) in T. nigroviridis has been cloned. Real-time fluorescence quantitative PCR showed that TnNLRP3 (T. nigroviridis NLRP3), TnASC (T. nigroviridis ASC) and Tncaspase-1 (T. nigroviridis caspase-1) mRNA in various tissues from health T. nigroviridis were highly expressed in immune-related tissues, such as spleen, gill, head kidney and intestine. After Vibrio parahemolyticus infection, the expression of TnNLRP3, TnASC and Tncaspase-1 mRNA in spleen, gill, head kidney reached a peak at 24 h, and the expression levels of these genes in intestine were the highest at 48 h. After the transfection of TnASC-pAcGFP-N1 monomer GFP plasmid into cos-7 cells, ASC specks, the activation marker of NLRP3 inflammasome, were observed. Bimolecular fluorescence complementarity and fluorescence colocation experiment showed that TnASC and Tncaspase-1 of TnNLRP3 inflammasome were co-located near the cell nucleus, and potentially interacted with each other. NLRP3 inflammasome inducer nigericin and agonist ATP could significantly induce the expression of TnNLRP3, TnASC and Tncaspase-1 mRNA, and activation of NLRP3 inflammasome could promote the generation of mature TnIL-1β (T. nigroviridis IL-1β). These results uncover that T. nigroviridis NLRP3 inflammasome could participate in the antibacterial immune response and the generation of mature TnIL-1β after activation.

Ammonia nitrogen stress increases susceptibility to bacterial infection via blocking IL-1R–Relish axis mediated antimicrobial peptides expression in shrimp

Ammonia nitrogen (ammonia-N) accumulation is pointed out as an environmental stress factor in the aquatic culture system, and has been shown to increase the susceptibility of aquatic organisms to infection. However, the mechanism by which ammonia-N stress induces a higher susceptibility to infection or disease outbreak remains largely unknown. Herein, we cloned and identified an interleukin-1 receptor homolog from the shrimp Litopenaeus vannamei (designated as LvIL-1R), and uncovered a LvIL-1R–LvRelish axis mediated antibacterial signaling that could induce the expression of antimicrobial peptides (AMPs) and thus conferred protection against Vibrio parahaemolyticus infection. Besides, we observed that shrimps became more susceptible to bacterial infection under ammonia-N stress, which attributed to that the LvIL-1R–LvRelish-AMPs antibacterial axis was restrained by the elevated ammonia-N concentration in hemolymph. Our results reveal how environmental (ammonia-N) stress increases shrimp susceptibility to infection, thereby providing some insights into the development of an effective environmental control strategy for disease prevention and treatment in aquaculture.

Effects of dietary yeast-derived nucleotide and RNA on growth performance, survival, immune responses, and resistance to Vibrio parahaemolyticus infection in Pacific white shrimp (Litopenaeus vannamei)

Nucleotides (NT) and RNA from yeast extracts are gaining interest as high-value feed additives. The present study intended to evaluate the influences of yeast derived-NT and RNA on the growth performance, survival, immune responses, and resistance to Vibrio parahaemolyticus infection in Pacific white shrimp. In Experiment 1, postlarvae were distributed into 7 groups, corresponding to 7 experimental diets: control, NT 0.25, NT 0.50, NT 0.75, RNA 0.25, RNA 0.50, and RNA 0.75 g/kg feed. They were fed the experimental diets for 45 days. Then, their body weights, survival rates, immune parameters, and Vibrio spp. counts in the hepatopancreas and intestines were determined. In Experiment 2, the shrimp from Experiment 1 were challenged by immersion with V. parahaemolyticus at 105 CFU/mL. Each group was fed the same diet for another 10 days to assess the disease resistance performance. The results revealed that the shrimp body weights of all groups were similar suggesting that neither NT nor RNA exerts the growth-promoting effect. However, the average survival rates of the NT and RNA groups were in the range of 89–93 %, significantly higher than that of the control (83 %). These increased survivals were in line with the reduction in the hepatopancreatic and intestinal Vibrio spp. counts and the elevated immune parameters in the NT and RNA-fed shrimp. At day 10 after the bacterial challenge, the highest survival rates were observed in the RNA 0.50 and 0.75 g/kg feed groups (81 % and 82 %, respectively), followed by the RNA 0.25 (70 %) and NT 0.75 g/kg feed (68 %), and significantly higher than the positive control (54 %). In short, both yeast-derived NT and RNA, especially the RNA at the dose of 0.50–0.75 g/kg feed groups, showed promising health benefits effects in the Pacific white shrimp, notably the improved immune function and disease resistance.

Identification and functional characterization of glutaredoxin 5 from the mud crab (Scylla paramamosain) in response to cadmium and bacterial challenge

Glutaredoxin (Grx) is a class molecule oxidoreductase, which plays a key role in maintaining redox homeostasis and regulating cell survival pathways. However, the expression pattern and function of Grx remain unknown in the mud crab (Scylla paramamosain). In the present study, a novel full-length of Grx 5 from the mud crab (designated as Sp-Grx 5) was cloned and characterized. The open reading frame of Sp-Grx 5 was 441 bp, which encoded a putative protein of 146 amino acids. The amino acid sequence of Sp-Grx 5 contained a typical C-G-F-S redox active motif and several GSH binding sites. Sp-Grx 5 widely existed in all tested tissues with a high-level expression in hepatopancreas. Subcellular localization showed that Sp-Grx 5 was located in the cytoplasm and nucleus. The expression of Sp-Grx 5 was significantly up-regulated after Vibrio parahaemolyticus infection and cadmium exposure, suggesting that Sp-Grx 5 was involved in innate immunity and detoxification. Furthermore, overexpression of Sp-Grx 5 could improve cells viability after H2O2 exposure. All these results indicated that Sp-Grx 5 played important roles in the redox homeostasis and innate immune response in crustaceans.

Effect of feeding different types of β-glucans derived from two marine diatoms (Chaetoceros muelleri and Thalassiosira weissflogii) on growth performance and immunity of banana shrimp (Penaeus merguiensis)

β-glucans are produced by many organisms and could be used as supplementary feed to enhance immunity and growth in some aquatic animals. This study aimed to compare the effectiveness of β-glucans derived from two marine diatoms (Chaetoceros muelleri and Thalassiosira weissflogii) as growth promoters and immunity enhancers in banana shrimp (Penaeus merguiensis). Shrimp were divided into 3 groups: the control group was fed without β-glucan; the second and the third group were fed with 2 g kg−1 of β-glucan derived from C. muelleri and T. weissflogii, respectively. Shrimp were fed over a 30-day period to determine growth performance (final weight, weight gain, average daily gain (ADG), and feed conversion ratio (FCR)) at day 15 and day 30, respectively. The immune parameters determined were total hemocyte count (THC), phenoloxidase activity (PO) and immune gene expression. Survival rates were measured after 14 days of the feeding trial and Vibrio parahaemolyticus infection (6, 24, 48 h post infection). There was no significant difference (P > 0.05) for growth stimulation of shrimps between the two types of β-glucans (C. muelleri or T. weissflogii). Notably, shrimps fed with β-glucans had a higher final weight, weight gain, and ADG (P < 0.05) than shrimps fed with the control diet, while FCR of shrimps fed with both β-glucans was lower when compared to the control diet. Immune parameters, THC, PO, and gene expression of anti-lipopolysaccharide factor (ALF) and crustin were significantly higher (P < 0.05) in shrimps fed with β-glucans, especially with β-glucans from C. muelleri than the control group both before and after V. parahaemolyticus infection. Expression of penaeidin 3 and peroxiredoxin genes was significantly higher in shrimps fed with β-glucans after bacterial infection. Histopathology of hepatopancreas revealed an increase in blasenzellen hepatopancreatic epithelial cells (B cells) after 14 days of feeding which remained higher following infection with V. parahaemolyticus. The survival rate of shrimps fed with the diet containing β-glucan derived from either C. muelleri (82.2%) or T. weissflogii (77.8%) after V. parahaemolyticus infection was significantly higher than for the control group (51.1%) (P < 0.05). In conclusion, we propose that feeding banana shrimps with β-glucans derived from marine diatoms either C. muelleri or T. weissflogii at a 2 g kg−1 diet can significantly improve their growth performance and immunity.

Integrative analyses of mRNA and microRNA expression profiles reveal the innate immune mechanism for the resistance to Vibrio parahaemolyticus infection in Epinephelus coioides

Vibrio parahaemolyticus, as one of the main pathogens of marine vibriosis, has brought huge losses to aquaculture. However, the interaction mechanism between V. parahaemolyticus and Epinephelus coioides remains unclear. Moreover, there is a lack of comprehensive multi-omics analysis of the immune response of grouper spleen to V. parahaemolyticus. Herein, E. coioides was artificially injected with V. parahaemolyticus, and it was found that the mortality was 16.7% in the early stage of infection, and accompanied by obvious histopathological lesions in the spleen. Furthermore, 1586 differentially expressed genes were screened by mRNA-seq. KEGG analysis showed that genes were significantly enriched in immune-related pathways, Acute-phase immune response, Apoptosis, Complement system and Cytokine-cytokine receptor interaction. As for miRNA-seq analysis, a total of 55 significantly different miRNAs were identified. Further functional annotation analysis indicated that the target genes of differentially expressed miRNAs were enriched in three important pathways (Phosphatidylinositol signaling system, Lysosome and Focal adhesions). Through mRNA-miRNA integrated analysis, 1427 significant miRNA–mRNA pairs were obtained and “p53 signaling pathway”, “Intestinal immune network for IgA production” were considered as two crucial pathways. Finally, miR-144-y, miR-497-x, novel-m0459-5p, miR-7133-y, miR-378-y, novel-m0440-5p and novel-m0084-3p may be as key miRNAs to regulate immune signaling pathways via the miRNA-mRNA interaction network. The above results suggest that the mRNA-miRNA integrated analysis not only sheds new light on the molecular mechanisms underlying the interaction between host and V. parahaemolyticus but also provides valuable and new insights into resistance to vibrio infection.

Quantitative Risk Assessment of Vibrio parahaemolyticus Toxi Infection Associated with the Consumption of Roasted Shrimp (Penaeus monodon)

In this study, a risk assessment on Vibrio parahaemolyticus infections was carried out in order to estimate the likelihood of gastroenteritis for Cameroonians after consumption of roasted shrimp (Penaeus monodon). The Codex Alimentarius Commission framework was used in this study. Based on the distribution of total V. parahaemolyticus in shrimp and literature information indicating that nonhaemolysing carrier strains could be pathogenic to humans, the cooking, and consumption patterns, the daily exposure level generated in this study, and the dose-response model from other studies, the infectious risk was evaluated and quantified by the Monte Carlo simulation. This simulation was realized based on 10,000 iterations using the Model Risk software, version 4.0, in combination with Microsoft Excel. To better quantify the exposure of consumers and the resulting risk of infection, several scenarios reflecting the minimal, average, and maximal exposures were undertaken. According to the results, the 90% confidence intervals for minimum and maximum exposures ranged from 15 to 24 colony-forming units per day (cells/day) and from 160 to 228 cells/day, respectively. Based on the modal scenario, 90% of the population consuming this shrimp is exposed to V. parahaemolyticus loads ranging from 74 to 110 cells/day, indicating a risk of infection ranging from 1.2 to 1.8 cases per million of consumption. The estimated number of annual disease cases based on annual production is between 1 and 10 cases. This reflects a relatively low risk of infection for roasted shrimp. Good hygiene practices during handling, cooking, and storage may help reduce the actual risk.

Identification and characterization of the novel bacteriophage BPVP-3325 for the biocontrol of Vibrio parahaemolyticus infection in seafood

A total of 19 bacteriophages were isolated from seawater, wet sand, sea rocks, and suspended solids collected from 10 different sites in Busan, Republic of Korea. Among them, the best-performed novel phage was designated as BPVP-3325 that demonstrated the highest lytic activity in 76% of the total studied strains of Vibrio parahaemolyticus. The complete genome of the novel phage was 222,634 bp with 42% G + C content; electron microscopy showed that the total length and head length were 411.66 ± 6.69 and 129.87 ± 5.10 nm, respectively. In vitro characterization, V. parahaemolyticus ATCC 17802 was used as a standard pathogenic strain of BPVP-3325 host. The latent period was approximately 40 min, and the burst size was 6.76 PFU/infected cell. BPVP-3325 demonstrated excellent efficacy in a relatively wide pH range of up to 5–10 and temperature of <50 °C. When ATCC 17802 was treated with BPVP-3325 in a liquid medium for 6 h, OD600 in the phage-treated group was significantly lower (~50%) than that in the host. The application of BPVP-3325 to oyster meat effectively inhibited the growth and activity of the standard strain (P < 0.05). Therefore, this study described the potential of novel BPVP-3325 therapy for natural oyster culture and food industry for the biocontrol of V. parahaemolyticus disease in aquaculture and human food.

Effects of replacing fish meal with cottonseed protein concentrate on the growth, immune responses, digestive ability and intestinal microbial flora in Litopenaeus vannamei

The effects of cottonseed protein concentrate (CPC) in place of fishmeal on the growth performance, immune response, digestive ability and intestinal microbiota of Litopenaeus vannamei were investigated in this study. L. vannamei (initial body weight: 0.42 ± 0.01g) was fed for 8 weeks by four isonitrogenous and isolipid feeds with CPC replacing fishmeal (FM) at 0% (control), 15% (CPC15), 30% (CPC30) and 45% (CPC45), respectively. At the end of the study, the final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR) and protein efficiency ratio (PER) of L. vannamei in CPC15 and CPC30 groups were significantly increased, while the feed conversion ratio (FCR) of L. vannamei in the CPC30 group was significantly reduced when compared with the FM group (P < 0.05). After Vibrio parahaemolyticus infection, the cumulative mortality of L. vannamei in CPC15 within 24 hpi was significantly lower than that of the control group (P < 0.05). When compared with the control group, the activities and expression of the immunity-related enzymes in the hepatopancreas had almost the same obvious change trend in the CPC-containing groups, which indicated that the replacement for fishmeal by CPC led to significant immune response in L. vannamei. Besides, significant up-regulation of the digestive enzyme activities were observed in the CPC-containing groups. Analysis of intestinal microbiota showed that significant difference in alpha diversity existed between the CPC-containing groups and the control group. The relative abundances of several top 10 dominated species at the phylum and genus levels were significantly changed in the CPC-containing groups compared with the control group (P < 0.05). Functional prediction of the microbiota indicated that the pathway of protein digestion and absorption was significantly more abundant while the pathways of nitrotoluene degradation, aminobenzoate degradation, atrazine degradation, dioxin degradation and xylene degradation were significantly less abundant in the CPC-containing groups than the FM group (P < 0.05). In summary, optimal dietary CPC replacement of FM could improve the growth, immunity, digestive capacity and the diversities of the intestinal microbial flora of L. vannamei. However, parts of the functions of the intestinal microbial flora were decline.

Transcriptome analysis reveals the function of TLR4-MyD88 pathway in immune response of Crassostrea hongkongensis against Vibrio Parahemolyticus

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.

Redclaw crayfish (Cherax quadricarinatus) responds to Vibrio parahaemolyticus infection by activating toll and immune deficiency signaling pathways and transcription of associated immune response genes

In invertebrates, several genes controlled by the Toll and immunodeficiency (IMD) signaling cascade are altered during microbial infection. However, little is known about the expression patterns of innate immune response genes in red-clawed crayfish (Cherax quadricarinatus). In the present study, the transcription of five genes was assessed in C. quadricarinatus challenged with Vibrio parahaemolyticus (V. parahaemolyticus). The expression of Relish, Toll-like receptor (TLR), tumor necrosis factor receptor-related factor 6 (TRAF6), Akirin, and IMD in different tissues and at different time points after infection were assessed. In addition, the Relish gene was amplified, the protein conformation of the Relish gene was predicted, and gene expression changes associated with antimicrobial peptide production in C. quadricarinatus were analyzed using RNA interference (RNAi). During V. parahaemolyticus infection, the transcripts of the above five genes were significantly increased in the hepatopancreas of C. quadricarinatus (P < 0.05). In contrast, TLR was significantly downregulated in muscle tissue at the initial stage of infection (P < 0.05); TRAF6 and IMD were significantly down-regulated throughout infection (P < 0.05); Akirin transcripts had the lowest abundance at 24 h post-infection; Relish, IMD and Akirin genes were significantly up-regulated in gill tissue at the early stage of infection (P < 0.05); only TRAF6 was significantly up-regulated at 6, 24 and 48 h after infection. The Relish gene of C. quadricarinatus is closely related to the Exopalaemon carinicauda. When the Relish gene was knocked down by RNAi, the V. parahaemolyticus challenge showed that the mortality rate of the RANi group was significantly higher than that of the NC group; pathological sections showed that the hepatopancreatic tissue damage was the most severe 12 h after the interference; and the interference significantly inhibited IRF4, NF-κB, ALF, laccase, SOD1, and lectin genes. Therefore, it can be hypothesized that the Toll and IMD pathways are activated in C. quadricarinatus in response to bacterial infection and that genes associated with these pathways are differentially transcribed in different tissues. This study provides insights into the Toll and IMD signaling pathways and the spatiotemporal expression of key genes regulating bacterial infection resistance in C. quadricarinatus.

Synergistic effects of dietary oxolinic acid combined with oxytetracycline on nonspecific immune responses and resistance against Vibrio parahaemolyticus infection of white shrimp (Penaeus vannamei)

This study investigated the synergistic effects of oxolinic acid (OA) combined with oxytetracycline (OTC) on white shrimp (Penaeus vannamei). Disk diffusion susceptibility testing was performed to analyze the sensitivity of Vibrio alginolyticus and Vibrio parahaemolyticus to different concentrations of OA and OTC. The results revealed that 50 mg OA/L combined with 50 mg OTC/L exhibited stronger antibacterial effects on V. alginolyticus and V. parahaemolyticus. The results of in vitro tests indicated that cotreatment with OA and OTC significantly reduced superoxide anion production and phenoloxidase activity, but not phagocytic activity. Subsequently, feeding trials were performed to investigate the immunomodulatory effects and bioaccumulation of dietary OA combined with OTC on shrimp. The healthy shrimp (15.13 ± 1.02 g) were divided into four groups: control, 100 mg OA/kg combined with 50 mg OTC/kg, 50 mg OA/kg combined with 100 mg OTC/kg, and 50 mg OA/kg combined with 50 mg OTC/kg. The shrimp were sampled to determine innate immunity parameters and residual OA and OTC levels in the muscle during a 28-day feeding regimen; the shrimp were fed the experimental diet from day 1 to day 5 and a commercial diet from day 6 to day 28. Residual OA levels were considerably higher in the group fed 100 mg OA/kg combined with 50 mg OTC/kg compared with the other groups and peaked on day 4. The residual OA levels of all the groups were below the detection limit after without providing OA. The residual OTC levels of the group fed 50 mg OA/kg combined with 100 mg OTC/kg were considerably higher from day 1 to day 4. The residual OTC levels in all the groups decreased rapidly and could not be detected on day 28. The administration of 50 mg OA/kg combined with 100 mg OTC/kg exerted the least effect on the white shrimp. Moreover, the survival rates of the treatment groups after the V. parahaemolyticus challenge were higher than those of the control group, especially the group fed 50 mg OA/kg combined with 100 mg OTC/kg. This result indicated that the synergistic effects of dietary OA and OTC are safe and effective. Combination therapy is a new method of antibiotic use in aquaculture.

Construction and immunological evaluation of live vector vaccine based on attenuated Listeria monocytogenes vector against Vibrio parahaemolyticus infection

Vibrio parahaemolyticus is considered to be one of the most threatening aquatic pathogens in aquaculture. Therefore, the development of an effective vaccine is crucial to prevent and control it. In this report, we identified the target antigen VP2309 with strong immunogenicity from four outer membrane proteins of V. parahaemolyticus. Furthermore, the live vector vaccine strain EGD-e△actA/inlB-VP2309 based on attenuated Listeria monocytogenes (Lm) was successfully constructed, consisting of secretory signal peptide of P60 protein, strong promoter Plmo2196 of Lm, and VP2309. It was able to express and secrete VP2309 efficiently. However, its expression retarded the growth of the host strain. The results of immune protection evaluation showed that both EGD-e△actA/inlB and EGD-e△actA/inlB-VP2309 strains reduced the mortality of zebrafish infected with V. parahaemolyticus. However, the vaccine strain carrying the VP2309 did not show better immune protection than the empty vector. Thus, we found that the attenuated Lm vector could offer a certain immune protection to zebrafish infected with V. parahaemolyticus, and is a potential vaccine vector that can be used in fish vaccine.

Determination of the efficacy of using a serine protease gene as a DNA vaccine to protect against Vibrio parahaemolyticus infection in Litopenaeus vannamei

Serine proteases are proteolytic enzymes that exhibit biological roles in many biological systems. Previously, a Vibrio parahaemolyticus serine protease was reported to be a virulence factor. Here, the serine protease gene of V. parahaemolyticus was investigated as a DNA vaccine against V. parahaemolyticus in Litopenaeus vannamei. The serine protease gene was mutated to replace the conserved residues His82, Asp131 and Ser231 with Gly, Asp and Pro, respectively. Then, a pcDNA3.1 vector to express mutVpSP (mutant serine protease) was constructed for in vitro and in vivo DNA vaccine investigation. In vivo mutVpSP transcriptional analysis revealed expression in various immunized white shrimp tissues, such as hemocytes, hepatopancreas, stomach, intestine, gills, and muscle. The efficiency of prevention of V. parahaemolyticus infection was investigated in vaccinated shrimp, and the lowest cumulative mortality percentage was 30%, while the control shrimp had a 60% cumulative mortality rate. The immune system was stimulated in shrimp vaccinated with the DNA vaccine. The mRNA expression of the shrimp immune-responsive genes phenoloxidase, peroxinectin and C-type lectin was significantly upregulated. Additionally, the humoral and cellular immune responses, including the PO, phagocytic, and encapsulation activities and nodule formation, were elevated. These results suggested that the serine protease could be a V. parahaemolyticus virulence determinant and that this DNA vaccine could be applied as an effective vaccine candidate for control of acute hepatopancreatic necrosis disease syndrome (AHPND) in shrimp.

Life history of oysters influences Vibrio parahaemolyticus accumulation in Pacific oysters (Crassostrea gigas).

Vibrio parahaemolyticus infection in humans is associated with raw oyster consumption. Evaluation of V. parahaemolyticus presence in oysters is of most interest because of the economic and public health issues that it represents. To explore V. parahaemolyticus accumulation and depuration in adult Crassostrea gigas, we developed a GFP-tagged V. parahaemolyticus strain (IFVp201-gfp+ ), as well as a rapid and efficient quantification method in C. gigas oysters haemolymph by flow cytometry. Impact of the life history of C. gigas on accumulation and depuration of V. parahaemolyticus IFVp201 was subsequently investigated. We found that naive oysters, i.e. grown in controlled facilities with UV treated seawater, accumulated significantly more IFVp201 than environmental oysters, i.e. grown in intertidal environment. We hypothesized that environmental oysters could have been immune primed, thus could limit V. parahaemolyticus accumulation. Meanwhile, both naive and environmental oysters had similar depuration rates.