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Antimicrobial peptides (AMPs) are short and positively charged peptides with broad-spectrum antimicrobial activities. AMPs have been investigated as potential antibiotic alternatives to improve growth performance and prevent pathogen infection in the poultry industry. The antimicrobial peptide tilapia piscidin 4 (TP4) was derived from Oreochromis niloticus, possesses antimicrobial activities and immunomodulatory properties, promotes intestinal health, and protects against pathogen infection. The codon-optimized sequence of TP4 was introduced into the pPICZαA vector and transformed into Pichia pastoris. Large-scale expression was induced following culture with methanol in a 500-liter fermenter. Freeze drying of fermented rTP4 broth and then rTP4 evaluation as a feed additive for Gallus gallus domesticus were performed. The in vitro antimicrobial activity of recombinant TP4 (rTP4) against gram-positive and gram-negative pathogens was evaluated. Evaluation of the effect of temperature on the antimicrobial activity of rTP4 showed its high stability at high temperatures. rTP4 significantly enhanced the phagocytic activity of macrophage cells, indicating that rTP4 has a remarkable ability to stimulate macrophages. rTP4 was used as a dietary supplement at 0.75, 1.5, 3.0, 6.0 and 12% in G. g. domesticus for five weeks, and growth performance, gut microbiota composition, and histology were assessed. The 3.0% rTP4 supplement group showed a significant increase in weight gain ratio and feed efficiency compared to those of the basal broiler diet group. Crude rTP4 was expressed by yeast to significantly promote growth efficiency and resistance against pathogens in G. g. domesticus, which could indicate its use as a suitable alternative to antibiotics as feed additives in the poultry industry.

In this study, we examined DNA sequences of bacteria isolated from goldfish intestine and identified the species in collaboration with the Food Industry Research & Development Institute of Taiwan. One bacterial species, Enterococcus avium, was selected for evaluation of its antimicrobial effects and probiotic function. The antimicrobial effects of feed containing E. avium were investigated in tilapia challenged with Streptococcus agalactiae. We found that E. avium inhibits a variety of pathogens, including gram-positive and gram-negative bacteria, with the strongest inhibition observed for Pseudomonas aeruginosa. Moreover, E. avium cells disrupted by sonication retained their antibacterial effects. By treating the sonicated cells at different temperatures or pH, we discovered that heating the cells above 75 °C resulted in the loss of their inhibitory effects. Moreover, E. avium pellets resuspended at pH 3.0 exhibited strong bacteriostatic activity, whereas E. avium pellets resuspended in solutions of pH > 10 had diminished antimicrobial activity. Tilapia fed once or twice a day with high concentrations (1 × 1010, 1 × 109, or 1 × 108 cfu/g) of sonicated or viable bacteria exhibited higher growth rates than both the control group and the experimental group fed 1 × 107 cfu/g. In addition, tilapia fed high concentrations of sonicated bacteria twice a day exhibited a higher survival rate than fish fed viable bacteria after a challenge with S. agalactiae.

Hepcidin regulates iron homeostasis and host-defense mechanisms, while the hepcidin-like protein, Tilapia hepcidin (TH)2-3, functions as an antimicrobial peptide (AMP). Since AMP dietary supplements may be used as alternatives to antibiotics in livestock, we tested the effects of recombinant (r)TH2-3 as a dietary supplement in grouper aquaculture. rTH2-3 was produced by a Pichia pastoris expression system and exhibited thermostability and broad-spectrum antimicrobial activity. The feed conversion ratio and feed efficiency were determined in Epinephelus lanceolatus (grouper) fed with rTH2-3-supplemented diet for 28 days. In addition, grouper showed enhanced superoxide dismutase (SOD) activity after rTH2-3 feeding compared to regular-diet-fed fish. Gut microbiota analysis revealed that microbial diversity was enhanced by feeding grouper with 1% rTH2-3. After challenging grouper with Vibrio alginolyticus, differential regulation of immune-related genes in the liver and spleen was observed between the TH2-3 and regular-diet groups, including for genes associated with antimicrobial and pro-inflammatory functions, complement components, and major histocompatibility complex (Mhc). These findings suggest that overall immunity was improved. Thus, our results suggest long-term supplementation with rTH2-3 may be beneficial for aquacultured grouper. The beneficial effects of the supplement are likely based on changes in the commensal microbial community as well as immunomodulation.

To study the biological role of tilapia piscidin 3 (TP3) in Streptococcus agalactiae infection in vivo, TP3/DsRed overexpressing transgenic zebrafish were generated. Under normal growth conditions, TP3/DsRed transgenic zebrafish exhibited an orange-red body color, without any other obvious abnormalities. However, when compared to wild type fish, TP3/DsRed transgenic zebrafish were resistant to S. agalactiae infection. After infection, the TP3 overexpressing fish exhibited higher expression of Toll-like receptor 4a (TLR4a), interleukin (IL)-10, IL-22, and C3b. Furthermore, TP3/DsRed transgenic zebrafish exhibited reduced induction of proinflammatory cytokines, including TNFα, IL-1β, IL-21, MyD88, and nuclear factor (NF)-κB. Taken together, our data show that TP3 overexpression in zebrafish can effectively suppress proinflammatory responses and enhance production of C3b. Together, these actions are conducive to the resolution of inflammation and bacterial clearance. We further postulate that TP3 may exert its anti-inflammatory effects by enhancing TLR4a-mediated negative regulation of NF-κB.

Streptococcus agalactiae infection is one of the most significant bacterial diseases in tilapia aquaculture. Identification of immune-related genes associated with Streptococcus agalactiae infection may provide a basis for breeding selection or therapeutics to augment disease resistance. Therefore, we utilized transcriptome profiling to study the host response in tilapia following Streptococcus agalactiae infection. Based on GO and KEGG enrichment analyses, we found that differentially expressed genes are widely involved in immune-related pathways, including the induction of antimicrobial peptides. Moreover, the main components of two immune-related pathways (Toll-like receptor signaling and leukocyte transendothelial migration) and four environmental information processing pathways (TNF, PI3K-Akt, Jak-STAT and MAPK) were identified. Finally, a time-course expression profile for several of the identified transcripts including tilapia piscidin 3 (TP3), tilapia piscidin 4 (TP4), TLR2, TLR5, MyD88, TRAF6, p38, and interleukin components was performed by qRT-PCR. Collectively, these results provide a starting point to study molecular mechanisms of tilapia immune response to Streptococcus agalactiae infection and may be applied as a basis for developing disease resistant strains by breeding selection.

Supplementing chicken feed with antibiotics can improve survival and prevent disease outbreaks. However, overuse of antibiotics may promote the development of antibiotic-resistant bacteria. Recently, antimicrobial peptides have been proposed as alternatives to antibiotics in animal husbandry. Here, we evaluate the effects of antimicrobial peptide, Epinephelus lanceolatus piscidin (EP), in Gallus gallus domesticus. The gene encoding EP was isolated, sequenced, codon-optimized and cloned into a Pichia pastoris recombinant protein expression system. The expressed recombinant EP (rEP) was then used as a dietary supplement for G. g. domesticus; overall health, growth performance and immunity were assessed. Supernatant from rEP-expressing yeast showed in vitro antimicrobial activity against Gram-positive and Gram-negative bacteria, according to an inhibition-zone diameter (mm) assay. Moreover, the antimicrobial peptide function of rEP was temperature independent. The fermentation broth yielded a spray-dried powder formulation containing 262.9 μg EP/g powder, and LC-MS/MS (tandem MS) analysis confirmed that rEP had a molecular weight of 4279 Da, as expected for the 34-amino acid peptide; the DNA sequence of the expression vector was also validated. We then evaluated rEP as a feed additive for G. g. domesticus. Treatment groups included control, basal diet and rEP at different doses (0.75, 1.5, 3.0, 6.0 and 12%). Compared to control, rEP supplementation increased G. g. domesticus weight gain, feed efficiency, IL-10 and IFN-γ production. Our results suggest that crude rEP could provide an alternative to traditional antibiotic feed additives for G. g. domesticus, serving to enhance growth and health of the animals.

Picornavirus is a highly contagious virus that usually infects cloven hoofed animals and causes foot-and-mouth disease. This disease is a major threat to livestock breeding worldwide and may lead to huge economic losses. Because effective vaccines or antiviral drugs remain unavailable, the search for new agents to combat FMDV infections is ongoing. Antimicrobial peptides are known to possess a broad range of biological activities, including antibacterial, antiviral, antitumor and immunomodulatory effects. In this work, we used a cell culture FMDV replication assay to evaluate several antimicrobial peptides for their ability to act as antiviral agents. We found that a synthesized form of the Epinephelus coioides antimicrobial peptide, epinecidin-1 (Epi-1), was effective at combatting FMDV. Epi-1 is known to have broad spectrum antimicrobial activity and low toxicity to normal eukaryotic cells, making it a good candidate for use as a therapeutic agent.The 50% cytotoxic concentration (CC50) for BHK-21 cells was 19.5 μg/ml for synthesized Epi-1, and the 50% effective concentration (EC50) for viral inhibition was 0.6 μg/ml. The selectivity index was 31.4, as calculated by the CC50/EC50 ratio. Furthermore, Epi-1 showed virucidal activity against FMDV at high concentrations. Interestingly, our data also showed that FMDV infection was most impaired when Epi-1 was treated at the time of viral adsorption. Taken together, our data show that Epi-1 may be a promising candidate for development as an anti-FMDV agent.

This study was designed to investigate the antimicrobial activity of two synthetic antimicrobial peptides from an aquatic organism, tilapia piscidin 3 (TP3) and tilapia piscidin 4 (TP4), in vitro and in a murine sepsis model, as compared with ampicillin, tigecycline, and imipenem. Mice were infected with (NDM-1)-producing K. pneumonia and multi-drug resistant Acinetobacter baumannii, and subsequently treated with TP3, TP4, or antibiotics for different periods of time (up to 168 h). Mouse survival and bacterial colony forming units (CFU) in various organs were measured after each treatment. Toxicity was determined based on observation of behavior and measurement of biochemical parameters. TP3 and TP4 exhibited strong activity against K. pneumonia and A. baumannii in vitro. Administration of TP3 (150 μg/mouse) or TP4 (50 μg/mouse) 30 min after infection with K. pneumonia or A. baumannii significantly increased survival in mice. TP4 was more effective than tigecycline at reducing CFU counts in several organs. TP3 and TP4 were shown to be non-toxic, and did not affect mouse behavior. TP3 and TP4 are able at potentiate anti-Acinetobacter baumannii or anti-Klebsiella pneumonia drug activity, reduce bacterial load, and prevent drug resistance, indicating their potential for use in combating multidrug-resistant bacteria.

To address the growing concern over antibiotic-resistant microbial infections in aquatic animals, we tested several promising alternative agents that have emerged as new drug candidates. Specifically, the tilapia piscidins are a group of peptides that possess antimicrobial, wound-healing, and antitumor functions. In this study, we focused on tilapia piscidin 3 (TP3) and TP4, which are peptides derived from Oreochromis niloticus, and investigated their inhibition of acute bacterial infections by infecting hybrid tilapia (Oreochromis spp.) with Vibrio vulnificus and evaluating the protective effects of pre-treating, co-treating, and post-treating fish with TP3 and TP4. In vivo experiments showed that co-treatment with V. vulnificus and TP3 (20 μg/fish) or TP4 (20 μg/fish) achieved 95.3% and 88.9% survival rates, respectively, after seven days. When we co-injected TP3 or TP4 and V. vulnificus into tilapia and then re-challenged the fish with V. vulnificus after 28 days, the tilapia exhibited survival rates of 35.6% and 42.2%, respectively. Pre-treatment with TP3 (30 μg/fish) or TP4 (20 μg/fish) for 30 minutes prior to V. vulnificus infection resulted in high survival rates of 28.9% and 37.8%, respectively, while post-treatment with TP3 (20 μg/fish or 30 μg/fish) or TP4 (20 μg/fish) 30 minutes after V. vulnificus infection yielded high survival rates of 33.3% and 48.9%. In summary, pre-treating, co-treating, and post-treating fish with TP3 or TP4 all effectively decreased the number of V. vulnificus bacteria and promoted significantly lower mortality rates in tilapia. The minimum inhibitory concentrations (MICs) of TP3 and TP4 that were effective for treating fish infected with V. vulnificus were 7.8 and 62.5 μg/ml, respectively, whereas the MICs of kanamycin and ampicillin were 31.2 and 3.91 μg/ml. The antimicrobial activity of these peptides was confirmed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM), both of which showed that V. vulnificus disrupted the outer membranes of cells, resulting in the loss of cell shape and integrity. We examined whether TP3 and TP4 increased the membrane permeability of V. vulnificus by measuring the fluorescence resulting from the uptake of 1-N-phenyl-naphthylamine (NPN). Treating fish with TP3 and TP4 under different pH and temperature conditions did not significantly increase MIC values, suggesting that temperature and the acid-base environment do not affect AMP function. In addition, the qPCR results showed that TP3 and TP4 influence the expression of immune-responsive genes, including interleukin (IL)-1β, IL-6, and IL-8. In this study, we demonstrate that TP3 and TP4 show potential for development as drugs to combat fish bacterial infections in aquaculture.

Vibrio vulnificus infection causes severe economic losses in Oreochromis niloticus aquaculture by inducing pro-inflammatory cytokines, that lead to inflammation and mortality. Omega-3 fatty acids, such as Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA), have been reported for their anti-inflammatory and antibacterial abilities in murine and zebrafish models. However, the anti-inflammatory and antibacterial functions of DHA and EPA in commercial aquaculture organisms such as Oreochromis niloticus remain unknown. The present study demonstrates antibacterial function and transcriptional modulation of inflammation-associated genes by DHA and EPA in Vibrio vulnificus infection in Oreochromis niloticus fish models. The administration of EPA or DHA improved the Oreochromis niloticus survival rate against Vibrio vulnificus infection. The induction of proinflammatory cytokines, Interleukin (IL)-1β, IL-6, Tumor necrosis factor (TNF)-α, and Toll-like receptor (TLR)-2 by Vibrio vulnificus was suppressed in fish that were administered DHA. Bacterial membrane disruption and the killing of Vibrio vulnificus by EPA and DHA was observed using SEM, TEM, and cytoplasm leakage studies. In silico analysis of the transcription profile in Ingenuity Pathway Analysis software showed that DHA may enhance anti-Vibrio vulnificus activity in Oreochromis niloticus via the activation of peroxisome proliferator-activated receptor α (PPARα) to inhibit nuclear factor kappa B and suppress hepatocyte nuclear factor 4 α (HNF4α). In summary, the results of the present study demonstrated that DHA and EPA reduce the severity of Vibrio vulnificus infection and increase the survival rate of Oreochromis niloticus.

A tilapia (Oreochromis niloticus) myosin light chain 3 (Mlc3) promoter region (~4.3 kb) was isolated and characterized. Sequence analysis of the clone revealed high similarity with a tilapia gene encoding the Mlc3 promoter region, exon 1, and intron 1. The clone contained several putative binding sequences for transcription factors, including MEF-2, MYOG, MyoD, PKNOX1, and AREB6. Deletion of a region of the tilapia Mlc3 promoter (801 to -3,881 bp) enhanced promoter activity, as determined by direct injection of a luciferase reporter construct into skeletal muscle of Archocentrus nigrofasciatus. These findings suggest that the region between -801 and -3,881 bp may contain negative regulatory elements. Stable germline transgenic strains of the ornamental fish species A. nigrofasciatus var. carrying the Taiwan coral red fluorescent protein (TcRFP) driven by the Mlc3 promoter were established. F1 adult transgenic A. nigrofasciatus var. exhibited brilliant pink fluorescence in skeletal muscles in the daylight. Therefore, our current study demonstrates the feasibility of using the tilapia Mlc3 promoter to drive fluorescence in new fish species, such as Perciformes.

Viperin is an anti-viral protein, induced by viral infection. In this study, we examined whether over-expression of viperin in fish muscle could inhibit bacterial growth. We first obtained the cDNA sequence of tilapia viperin, through RT-PCR-mediated cloning and sequencing. The cDNA sequence was similar to those of several fish viperins in GenBank, and it was predicted to encode the conserved domain of radical S-adenosylmethionine superfamily proteins. Phylogenetic analysis revealed that tilapia viperin was most closely related to viperin of Sciaenops ocellatus, Coreoperca kawamebari, and C. whiteheadi. Expression of tilapia viperin was significantly up-regulated in the kidney, liver, spleen, and gills upon challenge with lipopolysaccharide (LPS) and poly(I:C) in a time- and dose-dependent manner. Injection of Vibrio vulnificus (204) and Streptococcus agalactiae (SA47) bacteria into tilapia resulted in significant induction of viperin expression in the whole body, kidney, liver, and spleen. Electrotransfer of a viperin-expressing plasmid into zebrafish muscles decreased bacterial numbers and altered expression of immune-related genes. These data indicate that such altered expression may account for the improvement in bacterial clearance following electroporation of viperin, suggesting that fish viperin has antiviral and antibacterial activities.

Groupers bred for mariculture are of high economic value in Taiwan. Unfortunately, these fish are highly susceptible to bacterial infection, and the use of antibiotics to control disease has had the unintended consequence of generating antibiotic-resistant bacteria. Herein, we examined whether it is possible to enhance grouper immunity through application of Bacillus coagulans, a probiotic bacterial species. In order to study its effects on immunity, B. coagulans was isolated from lactobacillus mixtures and administered live to grouper (Epinephelus coioides) and zebrafish (Danio rerio). Fish were fed for 30 days on a diet consisting of B. coagulans (104, 106, 108, or 1010 colony-forming units (cfu) of B. coagulans in 50 ml of media) mixed with 50 g of eel powder. After 30 days, disease resistance against Vibrio vulnificus (204) infection and expression of immune-related genes were determined. We found that oral administration of B. coagulans significantly enhanced expression of several immune-related genes, including myeloid differentiation factor (MyD)88, interleukin (IL)-1β, tumor necrosis factor (TNF)-2, and IL-1β in grouper, and Toll-like receptor (TLR) 4, TNF-α, TRAM 1, and nuclear factor (NF)-κB in zebrafish. Treatment with B. coagulans significantly reduced mortality at 24, 48, 72, and 96 h after challenge with V. vulnificus (204). In vitro studies revealed that B. coagulans possesses bactericidal activity against Enterococcus faecalis (10066), Staphylococcus aureus subsp. (10780), Streptococcus agalactiae (S47, S48, 819), Escherichia coli (10675), Klebsiella oxytoca (13985), Pseudomonas aeruginosa (19660), and V. vulnificus (204, 12905, YJ016). On average, 107 colony-forming units of B. coagulans were required for bactericidal activity. In conclusion, our results indicate that dietary intake of B. coagulans enhances expression of immune-related genes and protects grouper and zebrafish against V. vulnificus (204) infection.

Micro (mi)RNAs are abundant small noncoding RNAs found in plants and animals, the regulatory functions of which are not fully understood in fish. To identify potential miRNAs, we screened an miRNA microarray with total RNA from zebrafish infected with Vibrio harveyi and another from uninfected zebrafish. Six miRNAs were obtained from the microarray screening. We studied miRNA expression patterns of 2 miRNAs (miR-122 and miR-194) after bacterial infection of transgenic zebrafish (containing tilapia hepcidin (TH)2-3) and non-transgenic zebrafish from which the 2 miRNAs were obtained from the microarray experiment. The results indicated that miR-122 and miR-194 were higher in PBS-injected zebrafish compared with TH2-3 zebrafish or wild-type (WT) zebrafish after V. harveyi infection. Overexpression of miRNAs (miR-122, miR-192, and miR-194a) was seen in zebrafish liver (ZFL) cells after lipopolysaccharide (LPS) treatment and in untreated fish. Our results showed that after 24 h of doxycycline treatment without LPS stimulation, interleukin (IL)-22, lysozyme, toll-like receptor (TLR)1, TLR3, TLR4a, and tumor necrosis factor (TNF)-α gene expressions were, respectively, upregulated by ~14-, 22-, 2.2-, 13-, 200-, and 38-fold in miR-122-transfected compared with non-transfected (WT) ZFL cells. In cells transfected with miR-192 and treated with LPS after 8–12 h, IL-22, lysozyme, TLR1, TLR3, TLR4a, and TNF-α expressions significantly differed between WT and miR-192-overexpressing ZFL cells. However, we observed significantly higher IL-22 expression levels after 12 h of LPS treatment in miR-192-transfected ZFL cells compared with non-transfected cells. In contrast, IL-22, lysozyme, and TNF-α were markedly upregulated (>100-fold) after miR-194a transfection and overexpression in ZFL cells and treatment with LPS. Our cloning and expression analyses indicated that miR-122, miR-192, and miR-194a play important roles in zebrafish immunology.

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.

We tested the activity of epinecidin-1, a novel antimicrobial peptide structurally related to pleurocidin, in commercial cleaning solutions stored at 4 and 25 degrees C for 7 and 14 days. The peptide's activities against Enterococcus faecalis, Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa, Staphylococcus aureus, Propionibacterium acnes, and Candida albicans were measured in a minimum inhibitory concentration (MIC) determination, minimal bactericidal concentration (MBC) determination, disk diffusion test, and a count of the bacterial numbers. Exposure to epinecidn-1 in a cleaning solution following MIC value comparisons in the disk diffusion test and counts of bacterial numbers after 16, 24, 48, and 72 h suggested that bacterial numbers were much lower than those treated with only commercial cleaning solutions for all bacteria. The efficacy of the antimicrobial activities of inhibiting bacterial numbers by epinecidin-1 in cleaning solutions at a low pH and a low temperature was not affected. Given its simple structure and antimicrobial activity, epinecidin-1 may be a useful component of microbicides designed to prevent pathogen infections and/or remediate abnormal vaginal or skin flora.

Artemia has been used extensively in aquaculture as fodder for larval fish, shrimp, and shellfish. Epinecidin-1, an antimicrobial peptide, was isolated from grouper (Epinephelus coioides) in 2005. Epinecidin-1 has been previously reported to possess antimicrobial activity against several Gram-positive and Gram-negative bacterial species, including Staphylococcus coagulase, Pseudomonas aeruginosa, Streptococcus pyogenes, and Vibrio vulnificus. In this study, we used electroporation to introduce plasmid DNA encoding a green fluorescent protein (EGFP)-epinecidin-1 fusion protein under the control of the cytomegalovirus (CMV) promoter into decapsulated Artemia cysts. Optimization of various properties (including cyst weight (0.2 g), plasmid concentration (50 μg/100 μl), and pulse voltage (150 V), length (10 ms), and number (2)) resulted in a hatching rate of 41.15%, a transfection efficiency of 49.81%, and a fluorescence intensity (A.U.) of 47.46. The expression of EGFP-epinecidin-1 was first detected by quantitative RT-PCR at 120 h post-electroporation, and protein was identified by Western blot at the same time. Furthermore, the EGFP-epinecidin-1 protein inhibited V. vulnificus (204) growth, as demonstrated by zone of inhibition studies. Zebrafish fed on transgenic Artemia expressing CMV-gfp-epi combined with commercial fodder were more resistant to infection by V. vulnificus (204): survival rate was enhanced by over 70% at 7, 14, and 21 days post-infection, and bacterial numbers in the liver and intestine were reduced. In addition, feeding of transgenic Artemia to zebrafish affected the immunomodulatory response to V. vulnificus (204) infection; expression of immune-responsive genes, including hepcidin and defbl2, was altered, as shown by qPCR. These findings suggest that feeding transgenic Artemia expressing CMV-gfp-epi to larval fish has antimicrobial effects, without the drawbacks of introducing drug residues or inducing bacterial drug resistance.

Piscidins are antimicrobial peptides (AMPs) that play important roles in helping fish resist pathogenic infections. Through comparisons of tilapia EST clones, the coding sequences of five piscidin-like AMPs (named TP1∼5) of Nile tilapia, Oreochromis niloticus, were determined. The complete piscidin coding sequences of TP1, -2, -3, -4, and -5 were respectively composed of 207, 234, 231, 270, and 195 bases, and each contained a translated region of 68, 77, 76, 89, and 64 amino acids. The tissue-specific, Vibrio vulnificus stimulation-specific, and Streptococcus agalactiae stimulation-specific expressions of TP2, -3, and -4 mRNA were determined by a comparative RT-PCR. Results of the tissue distribution analysis revealed high expression levels of TP2 mRNA in the skin, head kidneys, liver, and spleen. To study bacterial stimulation, S. agalactiae (SA47) was injected, and the TP4 transcript was upregulated by >13-fold (compared to the wild-type (WT) control, without injection) and was 60-fold upregulated (compared to the WT control, without injection) 24 h after the S. agalactiae (SA47) injection in the spleen and gills. Synthesized TP3 and TP4 peptides showed antimicrobial activities against several bacteria in this study, while the synthesized TP1, -2, and -5 peptides did not. The synthesized TP2, -3, and -4 peptides showed hemolytic activities and synthesized TP3 and TP4 peptides inhibited tilapia ovary cell proliferation with a dose-dependent effect. In summary, the amphiphilic α-helical cationic peptides of TP3 and TP4 may represent novel and potential antimicrobial agents for further peptide drug development.

Immunostimulatory effects of the oral administration of the recombinant epinecidin-1 protein from BL21 Escherichia coli (containing the pET28a-epinecidin-1-dsRed plasmid) were studied in grouper (Epinephelus coioides) and zebrafish (Danio rerio). For this purpose, fish were fed diets for 30 days containing the recombinant epinecidin-1 protein from BL21 E. coli (containing the pET28a-epinecidin-1-dsRed plasmid) at different bacterial numbers (10(4), 10(6), 10(8), and 10(10) colony-forming units (cfu) of BL21 E. coli in 50 ml of LB medium) mixed with 50 g of eel powder as fodder. After 30 days of feeding, immune-related gene expressions for bacterial-infection responses and disease resistance against Vibrio vulnificus (204) were determined. The V. vulnificus (204) injected into the fish abdominal cavity mimicked gram-negative bacterial infections in culture ponds. Experimental results assessed whether the recombinant epinecidin-1 protein from BL21 E. coli (containing the pET28a-epinecidin-1-dsRed plasmid) has up- (or down-) regulation immune-related genes expression. Results indicated that the recombinant epinecidin-1 protein from BL21 E. coli administered as a feed supplement significantly enhanced expressions several immune-related genes such as tumor necrosis factor (TNF)-1 in grouper and Toll-like receptor (TLR)4, interleukin (IL)-1β, nitric oxide synthase (NOS)2, and nuclear factor (NF)-κB in zebrafish. After being challenged with V. vulnificus (204) for 24, 48, 72, or 96 h, the percentage mortality was significantly reduced in treated fish, which indicated that the recombinant epinecidin-1 protein from BL21 E. coli administered as a feed supplement could bring about downregulation of TNF-1 expression and functioned like an antagonist for binding TLR4, which reduced the signal transduction pathway for inhibiting TNF and IL-1β expressions while reducing binding of the transcription factor, NF-κB, to TNF and the IL-1β promoter region. The experimental results indicated that dietary intake of the recombinant epinecidin-1 protein from BL21 E. coli modulated immune-related gene expressions and disease resistance of grouper and zebrafish after a V. vulnificus (204) infection.

In the present study, we used Vibrio vulnificus and a zebrafish model system to investigate the inhibitory effect of epinecidin-1 on acute bacterial infection and studied the impacts of pretreatment, co-treatment, and post-treatment with epinecidin-1 on its protective efficacy. In vivo experiments showed that co-treatment with epinecidin-1 and V. vulnificus achieved 78%-97% survival rates after 30 days. When epinecidin-1 and V. vulnificus were co-injected into zebrafish and zebrafish were re-challenged with V. vulnificus after 30 days, zebrafish had survival rates of 22%-47%. Pretreatment and post-treatment with epinecidin-1 obtained respective survival rates of 57% and 60%. In addition, epinecidin-1 modulated the expressions of immune-responsive genes like interleukin (IL)-10, IL-1b, tumor necrosis factor-α, and interferon-γ as analyzed by a microarray and qPCR approach. This study demonstrates the use of epinecidin-1 to develop inactivated material for fish bacterial infections which can provide guidelines for the future design of epinecidin-1-bacterial formulations for various in vivo applications.

The antimicrobial and immunomodulatory functions of the antimicrobial peptide, tilapia hepcidin (TH)2-3, against a bacterial endotoxin under in vitro conditions was previously reported. In this study, we investigated the antibacterial and immunomodulatory functions of TH2-3 in mice infected with the pathogen, Vibrio vulnificus. A TH2-3 injection in V. vulnificus-infected mice produced an increased survival rate compared to mice injected with V. vulnificus only. In addition, a TH2-3 injection increased the bacteriostatic property against V. vulnificus in mice. Gene expressions examined using a microarray demonstrated that TH2-3 modulated several V. vulnificus-responsive genes in the host. A neutralizing antibody assay of mice serum against inactivated V. vulnificus antigen-coated plates demonstrated the induction of an immune response by TH2-3 against the pathogen. Taken together, TH2-3 enhanced the survival rate of mice against the bacterial pathogen V. vulnificus through both antimicrobial and immunomodulatory functions. These properties make the TH2-3 peptide a good candidate for development as a new antimicrobial drug and suggest that TH2-3 can underpin the design of adjuvants for further development of vaccines.

The inhibitory function of tilapia hepcidin (TH)1-5, an antimicrobial peptide, was not examined in previous studies. In this study, we synthesized the TH1-5 peptide and tested TH1-5's antitumor activity against several tumor cell lines. We show that TH1-5 inhibited the proliferation of tumor cells and reduced colony formation in a soft agar assay. Scanning electron microscopy and transmission electron microscopy showed that TH1-5 altered the membrane structure similar to the function of a lytic peptide. Acridine orange/ethidium bromide staining, a wound-healing assay, and a flow cytometric analysis showed that TH1-5 induced necrosis with high-concentration treatment and induced apoptosis with low-concentration treatment. Inflammation is known to be closely associated with the development of cancer. TH1-5 showing anti-inflammatory effects in a previous publication induced us to evaluate the anti-inflammatory effects in cancer cell lines through the expressions of immune-related genes after being treated with the TH1-5 peptide. However, real-time qualitative RT-PCR indicated that TH1-5 treatment induced downregulation of the expressions of interleukin (IL)-6, IL-8, IL-12, IL-15, interferon-γ, CTSG, caspase-7, and Bcl-2, and upregulation of IL-2 and CAPN5 in HeLa cells, and upregulation of IL-8 and CTSG in HT1080 cells. These results suggest that TH1-5 possibly induces an inflammatory response in HeLa cells, but not in HT1080 cells. Overall, these results indicate that TH1-5 possesses the potential to be a novel peptide for cancer therapy.

Hepcidin is an antimicrobial peptide (AMP) secreted by the liver during inflammation that plays a central role in mammalian iron homeostasis. But the function of hepcidin in fish is still not completely understood. We recently described three different hepcidins (named tilapia hepcidin (TH)1-5, TH2-2, and TH2-3) from tilapia Oreochromis mossambicus, the cDNA sequences were determined, the predicted peptides were synthesized, and the TH2-3 peptide showed antimicrobial activity against several bacteria. We hypothesized that TH2-3 may have a biological function like an AMP in fishes and can be used as a transgene to boost resistance against bacterial infection. To examine the antimicrobial effects of TH2-3, we produced and engineered the overexpression of TH2-3 in zebrafish (Danio rerio) and the convict cichlid (Archocentrus nigrofasciatus). The microinjected plasmid also contained a green fluorescent protein (GFP) which was used as an indicator to trace germline transmission. In vivo, transgenic TH2-3 fish (of the F3 generation) were challenged with Vibrio vulnificus (204) and Streptococcus agalactiae (SA). Results showed significant clearance of bacterial numbers of V. vulnificus (204) but not of S. agalactiae in transgenic TH2-3 fish. A gene expression study using a real-time RT-PCR revealed that transgenic TH2-3 zebrafish showed increased endogenous expressions of Myd88, tumor necrosis factor-alpha, and TRAM1 in vivo. After transgenic TH2-3 zebrafish were infected with V. vulnificus (204), interleukin (IL)-10, IL-26, lysozyme, toll-like receptor (TLR)-4a, and Myd88 were upregulated, but IL-1beta (at 12-24 h) and IL-15 (at 1-12 h) were downregulated post-infection. After transgenic TH2-3 zebrafish were infected with S. agalactiae, IL-1beta (at 1-24 h), IL-15 (at 6 h), IL-22 (at 1-6 h), and TLR3 (at 1-24 h) were downregulated, but TLR4a (at 6-12 h) and c3b (at 12 h) were upregulated post-infection. Our findings identify the TH2-3 transgene in transgenic fish as an active component of the host response to bacterial pathogens. These results suggest that using TH2-3 as a transgene in zebrafish can effectively inhibit bacterial growth, specifically the V. vulnificus (204) strain for up to 24 h.

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)-kappaB activating protein-like were upregulated, but IL-1beta and tumor necrosis factor-alpha were downregulated at 12h post-infection; IL-21, complement component c3b, and NF-kappaB activating protein-like were downregulated, but MyD88 was upregulated at 24h post-infection. These results suggest that using epinecidin-1 as a transgene in zebrafish can effectively inhibit bacterial growth for up to 24h after infection.

The synthetic epinecidin-1(22-42) peptide was derived from positions 22-42 of Epinephelus coioides epinecidin-1. The synthetic SALF(55-76) cyclic peptide (csSALF(55-76)) and SALF(55-76) linear peptide (lsSALF(55-76)) contained sequences from positions 55 to 76 of the Penaeus monodon anti-lipopolysaccharide factor (ALF), respectively. We studied the in vitro activities of epinecidin-1(22-42), csSALF(55-76), and lsSALF(55-76) against Propionibacterium acnes, Candida albicans, and Trichomonas vaginalis. The minimum inhibitory concentrations (MICs) of epinecidin-1(22-42) for the test pathogen strains ranged 12.5-200 microg/ml, those of csSALF(55-76) ranged 100-200 microg/ml, and those of lsSALF(55-76) ranged 25-200 microg/ml. epinecidin-1(22-42) exhibited cytotoxicity towards P. acnes, C. albicans, and T. vaginalis (one strain of which was a metronidazole-resistant strain, while the other strain was not), suggesting that epinecidin-1 functions like a lytic peptide. Similar cytotoxicity was identified against T. vaginalis treated with the csSALF(55-76) and lsSALF(55-76) peptides. The antimicrobial activities of these peptides were confirmed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), a viable cell count assay, and flow cytometric analysis. TEM and SEM examinations of T. vaginalis treated with these three peptides showed that severe swelling preceded cell death and breakage of the outer membrane, and the intracellular inclusion was found to have effluxed extracellularly. This phenomenon was also found with epinecidin-1(22-42) treatment of P. acnes and C. albicans. Our results suggest that the epinecidin-1(22-42), csSALF(55-76), and lsSALF(55-76) peptides may be good candidates for treating trichomoniasis, and epinecidin-1(22-42) may have potential as a drug supporting therapy for acne and candidiasis.