Inactivation of nervous necrosis virus infecting grouper (Epinephelus coioides) by epinecidin-1 and hepcidin 1–5 antimicrobial peptides,and downregulation of Mx2 and Mx3 gene expressions

Betanodaviruses are one of the serious pathogens in nervous necrosis viral (NNV) disease that brings about mortality in the larval stage of grouper (Epinephelus coioides). In this study, the efficacy of pretreatment, co-treatment, and posttreatment with the antimicrobial epinecidin-1 and hepcidin 1–5 peptides against a betanodavirus was evaluated by intraperitoneal inoculation in grouper. The results showed that co-treatment of epinecidin-1 or hepcidin 1–5 with the virus was effective in promoting a significant decrease in grouper mortality. Re-challenge with virus again after 30 day in co-treated grouper groups showed high survival suggesting that epinecidin-1 and hepcidin 1–5 enhanced fish survival. However, grouper inoculated with NNV and then inoculated with epinecidin-1 8 h later showed significantly different survival from the group inoculated with virus alone, suggesting that epinecidin-1 can be used as a drug to rescue infected grouper. Infection after pretreatment, co-treatment, and posttreatment with epinecidin-1 or hepcidin 1–5 was verified by RT-PCR which showed downregulation of Mx2 and Mx3 gene expressions. All these data strongly suggest that epinecidin-1 and hepcidin 1–5 are effective peptides for protecting grouper larvae by reducing NNV infection.     

Transgenic expression of tilapia hepcidin 1-5 and shrimp chelonianin in zebrafish and their resistance to bacterial pathogens

Recently, tilapia hepcidin (TH)1-5 was characterized, and its antimicrobial functions against several pathogens were reported. The antimicrobial functions of another shrimp antimicrobial peptide (AMP), chelonianin, were also characterized using a recombinant chelonianin protein (rcf) that was expressed by a stably transfected Chinese hamster ovary (CHO) cell line against pathogen infections in fish. The function of the overexpression of both AMPs in zebrafish muscles was not examined in previous studies. Herein, we investigated the antimicrobial functions of TH1-5 and chelonianin against Vibrio vulnificus (204) and Streptococcus agalactiae (SA48) in transgenic TH1-5 zebrafish and transgenic chelonianin zebrafish. The presence of TH1-5 and chelonianin enhanced the inhibitory ability in transgenic AMP zebrafish against the two different bacterial infections. The bacterial number of either V. vulnificus (204) or S. agalactiae (SA48) had decreased at 96?h after injection into transgenic AMP zebrafish muscle compared to non-transgenic zebrafish muscle. Additionally, immune-related gene expressions analyzed by real-time PCR studies showed the modulation of several genes including interleukin (IL)-10, IL-22, IL-26, MyD88, Toll-like receptor (TLR)-1, TLR-3, TLR-4, nuclear factor (NF)-κB, tumor necrosis factor (TNF)-α, and lysozyme, and significant differences were found between transgenic AMP zebrafish and wild-type zebrafish injected with PBS at 1-24?h. These results suggest that several immune-related gene expressions were induced in transgenic TH1-5 and chelonianin zebrafish which effectively inhibited bacterial growth. The survival rate dropped to 86.6% in transgenic chelonianin zebrafish after 28 days of infection compared of the 50% survival rate in transgenic TH1-5 zebrafish after 28 days of infection. Overall, these results indicate that TH1-5 and chelonianin possess the potential to be novel candidate genes for aquaculture applications to treat fish diseases.     

Immune response and inhibition of bacterial growth by electrotransfer of plasmid DNA containing the antimicrobial peptide,epinecidin-1, into zebrafish muscle

Bacterial infections represent serious diseases in aquaculture, rapidly leading to fish death by septicemia. We investigated whether the electrotransfer of green fluorescent protein gene fusion epinecidin-1 (CMV-gfp-epi) DNA into zebrafish muscle could regulate the fish immune response and inhibit bacterial growth. Electroporation parameters such as the number of pulses, voltage, and amount of plasmid DNA were analyzed, and results demonstrated the greatest mRNA expression level of gfp-epi relative to β-actin was obtained with a pulse number of 4, a voltage strength of 100 V/cm, a concentration of DNA of 90 μg/fish, and electroporation for 96 h. In addition, the cytomegalovirus (CMV) promoter exhibited higher activity compared to the mylz promoter in muscle for electrotransfer in zebrafish. GFP fluorescence and gfp-epi mRNA expression in tissues after electroporation were also studied by a polymerase chain reaction, immunohistochemistry, and fluorescence microscopy. gfp-epi expression was significantly correlated with decreased bacterial numbers and immune-related gene expression. These data demonstrate that electroporation of epinecidin-1 might have provoked an inflammatory response that accounts for the improvement in bacterial clearance.     

The antimicrobial peptide, epinecidin-1, mediates secretion of cytokines in the immune response to bacterial infection in mice

Epinecidin-1, an antimicrobial peptide which encodes 21 amino acids, was isolated from a marine grouper (Epinephelus coioides). In this study, we investigated its immunomodulatory functions in mice co-injected with Pseudomonas aeruginosa. In vivo results showed that the synthetic epinecidin-1 peptide induced significant secretion of immunoglobulin G1 (IgG1) in mice co-injected with P. aeruginosa. Moreover, after injection of 40, 100, 200, or 500 μg epinecidin-1/mouse, we detected IgM, IgG, IgG1, and IgG2a in mice treated for 1, 2, 3, 7, 14, 21, and 28 days. Results showed that there were no significant differences in IgM, IgG, or IgG2a between mice injected with epinecidin-1 alone. IgG1 increased to a peak at 24 h, 7 days, and 28 days after an epinecidin-1 (40 μg/mouse) injection. Injection of 500 μg epinecidin-1/mouse increased IgG1 to peaks at 2 and 3 days; injection of 100 μg epinecidin-1/mouse increased IgG1 to a peak at 21 days. This supports epinecidin-1 being able to activate the Th2 cell response (enhance IgG1 production) against P. aeruginosa infection. Treatment with different concentrations of epinecidin-1 in mice elevated plasma interleukin (IL)-10 to initial peaks at 24 and 48 h, and it showed a second peak at 16 days. In RAW264.7 cells, treatment with epinecidin-1 alone did not produce significant changes in tumor necrosis factor (TNF)-α protein secretion at 1, 6, or 24h after treatment with 3.75, 7.5, or 15 μg/ml epinecidin-1 compared to the lipopolysaccharide group.     

Using an improved Tol2 transposon system to produce transgenic zebrafish with epinecidin-1 which enhanced resistance to bacterial infection

In order to advance the application of antimicrobial peptides in aquaculture, transgenic zebrafish expressing the antimicrobial peptide, epinecidin-1, were developed and are reported on here. First, we cloned the zebrafish mylz2 promoter for this purpose. To characterize the activity of the mylz2 promoter, various fragments of it were analyzed using a firefly luciferase transient expression assay, in which maximum promoter activity was found with a 2.5-kb fragment. In addition, the 2.5-kb fragment also expressed considerable red fluorescent proteins in skeletal muscles of transgenic zebrafish. Second, in order to improve the translation efficiency of the Tol2 transposase, we constructed untranslated regions (UTRs) of zebrafish ba1 globin flanked by a transposase. A transient embryonic excision assay (TEEA) and in vivo fluorescent observations showed high transposition efficiency during embryonic development. After optimization of the promoter and transgene efficiencies, transgenic zebrafish with the Epi-1/DsRed plasmid (pTLR-m2.5 K-K.Epinecidin-1/DsRed vector) were developed, and expressions of Epi-1/DsRed in muscles and blood were demonstrated by immunohistochemical staining techniques. Moreover, we also found that the Epi-1/DsRed gene was efficiently and significantly expressed in vivo against Vibrio vulnificus and Streptococcus agalactiae after injecting the bacteria and determining bacterial counts. A gene expression study using real-time RT-PCR revealed that Epi-1/DsRed itself induced endogenous MyD88 expression in vivo. After Epi-1/DsRed transgenic zebrafish were infected with V. vulnificus 204, interleukin (IL)-10, IL-22, IL-26, lysozyme, toll-like receptor (TLR)1, TLR3, TLR4a, MyD88, and nuclear factor (NF)-κB activating protein-like were upregulated, but IL-1β and tumor necrosis factor-α were downregulated at 12 h post-infection; IL-21, complement component c3b, and NF-κB activating protein-like were downregulated, but MyD88 was upregulated at 24 h post-infection. These results suggest that using epinecidin-1 as a transgene in zebrafish can effectively inhibit bacterial growth for up to 24 h after infection.     

Effects of temperature and salinity on the survival of Vibrio vulnificus in seawater and shellfish.

Sterilized seawater was used to assess the effects of temperature and salinity on the survival of Vibrio vulnificus. In the temperature range of 13 to 22 degrees C, numbers of V. vulnificus increased during the 6-day incubation. Temperatures outside this range reduced the time of V. vulnificus survival in sterile 10-ppt seawater. At these restrictive temperatures, V. vulnificus numbers were reduced by 90% after 6 days of incubation. Incubation between 0.5 and 10.5 degrees C demonstrated that V. vulnificus survives poorly below 8.5 degrees C. At salinities between 5 and 25 ppt and at 14 degrees C, V. vulnificus numbers actually increased or remained unchanged after 6 days of incubation. At salinities of 30, 35, and 38 ppt, numbers of V. vulnificus decreased 58, 88, and 83%, respectively. V. vulnificus could not be recovered from deionized water, indicating lysis. When a rifampin-resistant strain of V. vulnificus was used to inoculate sterilized and unsterilized seawater (20 ppt, 20 degrees C), numbers increased in sterile seawater but decreased to undetectable levels in 14 days in the unsterilized seawater, indicating that biological factors may play a role in the survival of V. vulnificus in the environment. Since our studies demonstrated sensitivity to low temperatures, the survival of V. vulnificus in naturally contaminated oysters at temperatures of 0, 2, and 4 degrees C was also determined. Numbers of endogenous V. vulnificus in oyster shellstock increased by more than 100-fold in shellstock stored at 30 degrees C but were reduced approximately 10- and 100-fold after 14 days at 2 to 4 degrees C and 0 degrees C, respectively. We conclude that both biological and physicochemical factors are important to the survival of V. vulnificus in the environment and that temperature is critical to controlling its growth in oyster shellstock.     

Proteomic and functional analysis of zebrafish after administration of antimicrobial peptide epinecidin-1

Antimicrobial peptides (AMPs) play important roles in innate immunity. One such AMP, epinecidin-1, exhibits antibacterial effects in zebrafish. In the current study, we aimed to identify the antimicrobial-associated proteins affected by epinecidin-1 treatment, and to unravel the underlying antimicrobial molecular mechanisms of epinecidin-1. We analyzed proteome changes in epinecidin-1-treated zebrafish using two-dimensional electrophoresis (2DE) coupled to mass spectrometry. Several differentially expressed proteins were identified, some of which were validated by real-time quantitative RT-PCR. The differentially expressed proteins were mapped onto Ingenuity Pathway Analysis canonical pathways, to construct a possible protein–protein interacting network regulated by epinecidin-1; this network suggested a potential role of epinecindin-1 in cytoskeletal assembly and organization. Our findings imply that epinecidin-1 may stabilize the cytoskeleton network in host cells, thereby promoting resistance to bacterial infection.     

Enzyme immunoassay for identification of Vibrio vulnificus in seawater, sediment, and oysters

Historically, methods used to identify Vibrio vulnificus in environmental samples have been inadequate because isolation and identification procedures are time-consuming and fail to separate V. vulnificus from other bacterial species. We describe an enzyme immunoassay (EIA) and culture techniques which identified V. vulnificus in seawater, sediment, and oysters. The EIA used monoclonal antibody FRBT37 to a species-specific epitope of V. vulnificus. No cross-reactions were observed among 72 non-V. vulnificus strains comprising 34 species and 15 genera. In field trials, the EIA identified correctly 99.7% of 348 biochemically confirmed V. vulnificus isolates. The epitope corresponding to FRBT37 was found in cells lysed by Triton X-100, deionized H2O, and ultrasonication but was not found in culture supernatants, indicating that its location was intracellular. In addition, electron micrographs of V. vulnificus labeled with FRBT37-biotin-avidin-gold showed that epitope FRBT37 reacted with fragments of lysed cells but not whole cells. FRBT37 was expressed when V. vulnificus was cultured in different growth media. The minimum level of detection of the EIA was approximately 2,000 V. vulnificus cells per EIA well. Epitope FRBT37 was labile at 70 degrees C for 30 min. Immunoblot and EIA plate formats reduced assay time and facilitated handling large numbers of test samples.     

Enzyme immunoassay for identification of Vibrio vulnificus in seawater, sediment, and oysters.

Historically, methods used to identify Vibrio vulnificus in environmental samples have been inadequate because isolation and identification procedures are time-consuming and fail to separate V. vulnificus from other bacterial species. We describe an enzyme immunoassay (EIA) and culture techniques which identified V. vulnificus in seawater, sediment, and oysters. The EIA used monoclonal antibody FRBT37 to a species-specific epitope of V. vulnificus. No cross-reactions were observed among 72 non-V. vulnificus strains comprising 34 species and 15 genera. In field trials, the EIA identified correctly 99.7% of 348 biochemically confirmed V. vulnificus isolates. The epitope corresponding to FRBT37 was found in cells lysed by Triton X-100, deionized H2O, and ultrasonication but was not found in culture supernatants, indicating that its location was intracellular. In addition, electron micrographs of V. vulnificus labeled with FRBT37-biotin-avidin-gold showed that epitope FRBT37 reacted with fragments of lysed cells but not whole cells. FRBT37 was expressed when V. vulnificus was cultured in different growth media. The minimum level of detection of the EIA was approximately 2,000 V. vulnificus cells per EIA well. Epitope FRBT37 was labile at 70 degrees C for 30 min. Immunoblot and EIA plate formats reduced assay time and facilitated handling large numbers of test samples.     

Development and validation of a predictive model for the growth of Vibrio vulnificus in postharvest shellstock oysters

Postharvest growth of Vibrio vulnificus in oysters can increase risk of human infection. Unfortunately, limited information is available regarding V. vulnificus growth and survival patterns over a wide range of storage temperatures in oysters harvested from different estuaries and in different oyster species. In this study, we developed a predictive model for V. vulnificus growth in Eastern oysters (Crassostrea virginica) harvested from Chesapeake Bay, MD, over a temperature range of 5 to 30°C and then validated the model against V. vulnificus growth rates (GRs) in Eastern and Asian oysters (Crassostrea ariakensis) harvested from Mobile Bay, AL, and Chesapeake Bay, VA, respectively. In the model development studies, V. vulnificus was slowly inactivated at 5 and 10°C with average GRs of -0.0045 and -0.0043 log most probable number (MPN)/h, respectively. Estimated average growth rates at 15, 20, 25, and 30°C were 0.022, 0.042, 0.087, and 0.093 log MPN/h, respectively. With respect to Eastern oysters, bias (B(f)) and accuracy (A(f)) factors for model-dependent and -independent data were 1.02 and 1.25 and 1.67 and 1.98, respectively. For Asian oysters, B(f) and A(f) were 0.29 and 3.40. Residual variations in growth rate about the fitted model were not explained by season, region, water temperature, or salinity at harvest. Growth rate estimates for Chesapeake Bay and Mobile Bay oysters stored at 25 and 30°C showed relatively high variability and were lower than Food and Agricultural Organization (FAO)/WHO V. vulnificus quantitative risk assessment model predictions. The model provides an improved tool for designing and implementing food safety plans that minimize the risk associated with V. vulnificus in oysters.     

Influence of aquatic microbiota on the survival in water of the human and eel pathogen Vibrio vulnificus serovar E

The eel and human pathogen Vibrio vulnificus serovar E (biotype 2) is seldom isolated from natural waters, although it can survive in sterilized artificial seawater microcosms for years. The main objective of the present study was to investigate whether aquatic microbiota can limit its survival and recovery from water samples. A set of preliminary experiments of survival in microcosms containing natural seawater and water from eel farms showed that the persistence of this pathogen was mainly controlled by grazing, and secondarily by bacterial competition. The bacterial competition was further analysed in artificial seawater microcosms co-inoculated with selected virulent serovar E (VSE) strains and potential competitors. Competitors included V. vulnificus biotype 1 isolates and strains of selected species that can grow on the selective media designed for V. vulnificus isolation from water samples. Evidences of bacterial competition that was detrimental for VSE recovery were recorded. Thus, some species produced a deleterious effect on VSE strains under starvation, and others were able to use the resources more efficiently under nutrient input. These results suggest that an overgrowth of more efficient competitor bacteria in conventional media used for isolation of V. vulnificus could mask the recovery of VSE strains and explain the scarcity of reports on the isolation of this human and eel pathogen from natural waters.     

Validation of a green fluorescent protein-labeled strain of Vibrio vulnificus for use in the evaluation of postharvest strategies for handling of raw oysters

In this paper we describe a biological indicator which can be used to study the behavior of Vibrio vulnificus, an important molluscan shellfish-associated human pathogen. A V. vulnificus ATCC 27562 derivative that expresses green fluorescent protein (GFP) and kanamycin resistance was constructed using conjugation. Strain validation was performed by comparing the GFP-expressing strain (Vv-GFP) and the wild-type strain (Vv-WT) with respect to growth characteristics, heat tolerance (45 degrees C), freeze-thaw tolerance (-20(o) and -80 degrees C), acid tolerance (pH 5.0, 4.0, and 3.5), cold storage tolerance (5 degrees C), cold adaptation (15 degrees C), and response to starvation. Levels of recovery were evaluated using nonselective medium (tryptic soy agar containing 2% NaCl) with and without sodium pyruvate. The indicator strain was subsequently used to evaluate the survival of V. vulnificus in oysters exposed to organic acids (citric and acetic acids) and various cooling regimens. In most cases, Vv-GFP was comparable to Vv-WT with respect to growth and survival upon exposure to various biological stressors; when differences between the GFP-expressing and parent strains occurred, they usually disappeared when sodium pyruvate was added to media. When V. vulnificus was inoculated into shellstock oysters, the counts dropped 2 log(10) after 11 to 12 days of refrigerated storage, regardless of the way in which the oysters were initially cooled. Steeper population declines after 12 days of refrigerated storage were observed for both iced and refrigerated products than for slowly cooled product and product held under conservative harvest conditions. By the end of the refrigeration storage study (22 days), the counts of Vv-GFP in iced and refrigerated oysters had reached the limit of detection (10(2) CFU/oyster), but slowly cooled oysters and oysters stored under conservative harvest conditions still contained approximately 10(3) and >10(4) CFU V. vulnificus/oyster by day 22, respectively. The Vv-GFP levels in the oyster meat remained stable for up to 24 h when the meat was exposed to acidic conditions at various pH values. Ease of detection and comparability to the wild-type parent make Vv-GFP a good candidate for use in studying the behavior of V. vulnificus upon exposure to sublethal stressors that might be encountered during postharvest handling of molluscan shellfish.     

Protective mechanism of curcumin against Vibrio vulnificus infection

Curcumin, a natural polyphenolic flavonoid extracted from the rhizome of Curcuma longa L., has many beneficial biological activities. However, there are relatively few reports of the effects of curcumin on pathogen infections. This study examined the effect of curcumin on a Vibrio vulnificus infection. The cytotoxicity of V. vulnificus to HeLa cells was significantly inhibited by curcumin (at 10 or 30?μM). To further examine the inhibitory mechanism of curcumin against V. vulnificus-mediated cytotoxicity, the level of bacterial growth, bacterial motility, cell adhesion, RTX toxin expression and host cell reactions were evaluated. Curcumin inhibited V. vulnificus growth in HI broth. Curcumin inhibited both bacterial adhesion and RTX toxin binding to the host cells, which can be considered the major protective mechanisms for the decrease in V. vulnificus cytotoxicity. Curcumin also inhibited host cell rounding and actin aggregation, which are the early features of cell death caused by V. vulnificus. In addition, curcumin decreased the V. vulnificus-induced NF-κB translocation in HeLa cells. Finally, curcumin protected mice from V. vulnificus-induced septicemia. In conclusion, curcumin may be an alternative antimicrobial agent against fatal bacterial infections.     

Effect of silymarin on curcumin-induced mortality in zebrafish (Danio rerio) embryos and larvae

In presence of 7.5 microM of curcumin, no embryos or larva of zebrafish survived 3 days of incubation; however, coincubation with 144 microg/ml silymarin increased the survival rates of curcumin-treated embryos and larvae to about 70%. Moreover, in presence of 12.5 microM curcumin, all embryos died after 2 days of incubation; however, co-treatment with 144 microg/ml silymarin increased the survival rates of curcumin-treated embryos and larvae up to 60 and 50%, respectively. This protective effect was not found in the other phenolic compounds viz., ferulic acid, naringin, or crocin, tested. Finally, using a fluorescence microscope, accumulation of less curcumin has observed in the edema sac area of the larvae co-treated with curcumin and silymarin than in the larvae treated with curcumin only. The result suggests that the protective effects of silymarin may be due to a decreased accumulation of curcumin in the fish body.     

Survival of Vibrio vulnificus in shellstock and shucked oysters (Crassostrea gigas and Crassostrea virginica) and effects of isolation medium on recovery.

When two species of shellstock oysters were artificially contaminated with Vibrio vulnificus, the bacterium survived when the oysters were stored at 10 degrees C and below. Large numbers of endogenous V. vulnificus cells were found after 7 days at both 0.5 and 10 degrees C in uninoculated control oysters (Crassostrea virginica). Oysters allowed to take up V. vulnificus from seawater retained the bacterium for 14 days at 2 degrees C. The presence of V. vulnificus in the drip exuded from the shellstock presented a possibility of contamination of other shellstock in storage. V. vulnificus injected into shucked Pacific (Crassostrea gigas) and Eastern (C. virginica) oysters survived at 4 degrees C for at least 6 days. An 18-h most-probable-number enrichment step in alkaline peptone water gave higher recovery levels of V. vulnificus than did direct plating to selective agars. The survival of this pathogen in both shellstock and shucked oysters suggests a potential for human illness, even though the product is refrigerated.