Feeding olive flounder (Paralichthys olivaceus) with Lactococcus lactis BFE920 expressing the fusion antigen of Vibrio OmpK and FlaB provides protection against multiple Vibrio pathogens: A universal vaccine effect.

Vibriosis, an illness caused by the Vibrio bacteria species, results in significant economic loss in olive flounder farms. Here we present a novel anti-Vibrio feed vaccine protecting multiple strains of Vibrio pathogens, a universal vaccine effect. The vaccine was generated by engineering Lactococcus lactis BFE920 to express the fusion antigens of Vibrio outer membrane protein K (OmpK) and flagellin B subunit (FlaB). These antigen genes are highly conserved among Vibrio species. Olive flounder (7.1 ± 0.8 g and 140 ± 10 g) were fed the vaccine adsorbed to a regular feed (1 × 107 CFU/g) for one week with a 1-week interval, repeating three times (a triple boost). The vaccinated fish increased the significant levels of antigen-specific antibodies, T cell numbers (CD4-1, CD4-2, and CD8α), cytokine production (T-bet and IFN-γ), and innate immune responses (TLR5M, IL-1ß, and IL-12p40). Also, the survival rates of adult and juvenile fish fed the vaccine were significantly elevated when challenged with V. anguillarum, V. alginolyticus, and V. harveyi. In addition, weight gain rate and feed conversion ratio were improved in vaccinated fish. The feed vaccine protected multiple Vibrio pathogens, a universal vaccine effect, by activating innate and adaptive immune responses. This oral vaccine may be developed as an anti-Vibrio vaccine to protect against a broad spectrum of Vibrio pathogens.

A Lactococcus lactis BFE920 feed vaccine expressing a fusion protein composed of the OmpA and FlgD antigens from Edwardsiella tarda was significantly better at protecting olive flounder (Paralichthys olivaceus) from edwardsiellosis than single antigen vaccines.

Edwardsiellosis is a major fish disease that causes a significant economic damage in the aquaculture industry. Here, we assessed vaccine efficacy after feeding oral vaccines to olive flounder (Paralichthys olivaceus), either L. lactis BFE920 expressing Edwardsiella tarda outer membrane protein A (OmpA), flagellar hook protein D (FlgD), or a fusion antigen of the two. Feed vaccination was done twice with a one-week interval. Fish were fed regular feed adsorbed with the vaccines. Feed vaccination was given over the course of one week to maximize the interaction between the feed vaccines and the fish intestine. Flounder fed the vaccine containing the fusion antigen had significantly elevated levels T cell genes (CD4-1, CD4-2, and CD8α), type 1 helper T cell (Th1) subset indicator genes (T-bet and IFN-γ), and antigen-specific antibodies compared to the groups fed the single antigen-expressing vaccines. Furthermore, the superiority of the fusion vaccine was also observed in survival rates when fish were challenged with E. tarda: OmpA-FlgD-expressing vaccine (82.5% survival); FlgD-vaccine (55.0%); OmpA-vaccine (50%); WT L. lactis BFE920 (37.5%); Ctrl (10%). In addition, vaccine-fed fish exhibited increased weight gain (∼20%) and a decreased feed conversion ratio (∼20%) during the four week vaccination period. Flounder fed the FlgD-expressing vaccine, either the single or the fusion form, had significantly increased expression of TLR5M, IL-1ß, and IL-12p40, suggesting that the FlgD may be a ligand of olive flounder TLR5M receptor or closely related to the TLR5M pathway. In conclusion, the present study demonstrated that olive flounder fed L. lactis BFE920 expressing a fusion antigen composed of E. tarda OmpA and FlgD showed a strong protective effect against edwardsiellosis indicating this may be developed as an E. tarda feed vaccine.

Pellet feed adsorbed with the recombinant Lactococcus lactis BFE920 expressing SiMA antigen induced strong recall vaccine effects against Streptococcus iniae infection in olive flounder (Paralichthys olivaceus).

The aim of this study was to develop a fish feed vaccine that provides effective disease prevention and convenient application. A lactic acid bacterium (LAB), Lactococcus lactis BFE920, was modified to express the SiMA antigen, a membrane protein of Streptococcus iniae. The antigen was engineered to be expressed under the nisin promoter, which is induced by nisin produced naturally by the host LAB. Various sizes (40 ± 3.5 g, 80 ± 2.1 g, and 221 ± 2.4 g) of olive flounder (Paralichthys olivaceus) were vaccinated by feeding the extruded pellet feed, onto which the SiMA-expressing L. lactis BFE920 (1.0 × 10(7) CFU/g) was adsorbed. Vaccine-treated feed was administered twice a day for 1 week, and priming and boosting were performed with a 1-week interval in between. The vaccinated fish had significantly elevated levels of antigen-specific serum antibodies and T cell marker mRNAs: CD4-1, CD4-2, and CD8a. In addition, the feed vaccine significantly induced T cell effector functions, such as the production of IFN-γ and activation of the transcription factor that induces its expression, T-bet. When the flounder were challenged by intraperitoneal infection and bath immersion with S. iniae, the vaccinated fish showed 84% and 82% relative percent survival (RPS), respectively. Furthermore, similar protective effects were confirmed even 3 months after vaccination in a field study (n = 4800), indicating that this feed vaccine elicited prolonged duration of immunopotency. In addition, the vaccinated flounder gained 21% more weight and required 16% less feed to gain a unit of body weight compared to the control group. The data clearly demonstrate that the L. lactis BFE920-SiMA feed vaccine has strong protective effects, induces prolonged vaccine efficacy, and has probiotic effects. In addition, this LAB-based fish feed vaccine can be easily used to target many different pathogens of diverse fish species.

Distinct immune tones are established by Lactococcus lactis BFE920 and Lactobacillus plantarum FGL0001 in the gut of olive flounder (Paralichthys olivaceus).

The immune tone is defined as an immunological state during which the readiness for immune response is potentiated. The establishment of immune tone in the gut of olive flounder (Paralichthys olivaceus) was investigated by feeding Lactococcus lactis BFE920 (LL) or Lactobacillus plantarum FGL0001 (LP). LL-fed flounder showed significantly increased levels of regulatory genes (FOXP3, IL-10, and TGF-ß1), CD18, and CD83 in the gut. In contrast, LP feeding drastically increased proinflammatory genes (T-bet, IL-1ß, and IFN-γ) and CD18. This indicates that LL and LP establish different types of local immune tones in the gut through differential activation of innate immune cells: LL activates both macrophages and dendritic cells while LP activates macrophages only. Both of the immune tones required at least a total of 6 probiotic feeds during 72 h for a stable establishment. Once established, the type of immune tone remained steady even up to 30 days (a total of 60 feeds) probiotics feeding. The LL-induced regulatory immune tone enhanced the level of occludin, a tight junction molecule, significantly more than that observed with the proinflammatory immune tone established by LP feeding. Consequently, LL-fed fish showed considerably lower gut permeability than that of the LP-fed group. Furthermore, when orally challenged by Edwardsiella tarda, LL-fed flounder survived at a significantly higher rate than LP-fed fish. The data clearly demonstrate that individual probiotics establish distinct types of immune tone in the fish gut, which in turn influences the immunological status as well as the physiology of the gut. Selection of proper probiotics may be essential for optimal effects in aquaculture farming.

The effects of combined dietary probiotics Lactococcus lactis BFE920 and Lactobacillus plantarum FGL0001 on innate immunity and disease resistance in olive flounder (Paralichthys olivaceus).

The effects of a dietary probiotic mixture containing Lactococcus (Lc.) lactis BFE920 isolated from bean sprout and autochthonous Lactobacillus (Lb.) plantarum FGL0001 originally isolated from the hindgut of olive flounder (Paralichthys olivaceus) were investigated for the purpose of improving the probiotic effects of Lc. lactis BFE920 on the olive flounder. The immunostimulatory, disease protective, and weight gain effects of Lc. lactis BFE920 were significantly improved when olive flounder (average weight 37.5±1.26 g) were fed the probiotic mixture (log10 7.0 CFU each/g feed pellet) for 30 days. Flounder fed the mixture showed improved skin mucus lysozyme activity and phagocytic activity of innate immune cells compared to flounder fed a single probiotic agent or a control diet. While the levels of neutrophil activity in flounder fed the single probiotic agent or the mixture were similar, they were significantly higher than levels in a control group. Additionally, probiotic-fed flounder showed significantly increased expressions of IL-6, IL-8, and TNF-α in the intestine compared to the control group. Following a 30-day period of being fed probiotics or a control diet, the olive flounder were challenged with an i.p. injection of Streptococcus iniae (log10 6.0 CFU/fish). The groups fed the mixed probiotics, Lc. lactis BFE920, Lb. plantarum FGL0001, and the control diet had survival rates of 55%, 45%, 35%, and 20%, respectively. Flounder fed the probiotic mixture gained 38.1±2.8% more body weight compared to flounder fed the control diet during the 30-day study period. These data strongly suggest that a mixture of Lc. lactis BFE920 and Lb. plantarum FGL0001 may serve as an immunostimulating feed additive useful for disease protection in the fish farming industry.

Prebiotics as immunostimulants in aquaculture: a review.

Prebiotics are indigestible fibers that increase beneficial gut commensal bacteria resulting in improvements of the host's health. The beneficial effects of prebiotics are due to the byproducts generated from their fermentation by gut commensal bacteria. In this review, the direct effects of prebiotics on the innate immune system of fish are discussed. Prebiotics, such as fructooligosaccharide, mannanoligosaccharide, inulin, or ß-glucan, are called immunosaccharides. They directly enhance innate immune responses including: phagocytic activation, neutrophil activation, activation of the alternative complement system, increased lysozyme activity, and more. Immunosaccharides directly activate the innate immune system by interacting with pattern recognition receptors (PRR) expressed on innate immune cells. They can also associate with microbe associated molecular patterns (MAMPs) to activate innate immune cells. However, the underlying mechanisms involved in innate immune cell activation need to be further explored. Many studies have indicated that immunosaccharides are beneficial to both finfish and shellfish.

Study of the soothing effects of troxerutin in alleviating skin sensitivity.

BACKGROUND: Transient receptor potential vanilloid 1 (TRPV1) is associated with skin sensitivity and mainly activated by capsaicin and heat. Interestingly, troxerutin can inhibit TRPV1 activation. However, its efficacy in reducing skin sensitivity remains undetermined. AIMS: We evaluated the efficacy of troxerutin in alleviating skin sensitivity using clinical tests and in vitro experiments. METHODS: For the in vitro experiment, HaCaT keratinocytes were pretreated with different concentrations of troxerutin, followed by incubation with 50 µM capsaicin for 1, 24, or 48 h. The gene and protein expressions of four inflammatory cytokines involved in skin irritation were determined. Among 35 Korean women with sensitive skin recruited for the clinical trial, 13 were involved in assessing the immediate soothing effects of 0.1% and 0.0095% troxerutin following capsaicin irritation, whereas 22 participated in evaluating the preventive soothing effect of 10% and 1% troxerutin over 4 weeks against capsaicin- and heat-induced irritation. We evaluated the soothing rate using skin redness, visual analog scale, and high temperature sensitive index as evaluation indices. RESULTS: Troxerutin inhibited the mRNA and protein expressions of cytokines in capsaicin-treated keratinocytes. In the clinical study, 0.1% and 0.0095% troxerutin promptly alleviated capsaicin-induced skin redness, whereas 10% troxerutin notably decreased both the visual analog scale and high temperature sensitive index for capsaicin- and heat-related irritation. However, 1% troxerutin was only effective in reducing the visual analog scale in response to capsaicin irritation. CONCLUSIONS: Troxerutin can inhibit TRPV1 activation in clinical and in vitro tests.

Lactococcus lactis BFE920 activates the innate immune system of olive flounder (Paralichthys olivaceus), resulting in protection against Streptococcus iniae infection and enhancing feed efficiency and weight gain in large-scale field studies.

The protective effect of a food-grade lactic acid bacterium Lactococcus lactis BFE920 against disease of olive flounder (Paralichthys olivaceus) cultivated on a large scale was studied. Initially, antimicrobial activity of L. lactis against several fish pathogens was evaluated in vitro; the probiotic showed strong antibacterial activity against Streptococcus iniae, Streptococcus parauberis and Enterococcus viikkiensis, and moderate activity against Lactococcus garviae. When olive flounders were fed for two weeks with experimental diets containing varying concentrations of L. lactis (1 × 10(6), 5 × 10(6), 2.5 × 10(7) and 1.25 × 10(8) CFU/g feed), all the experimental feed groups showed 68-77% survival upon challenge with S. iniae. A field-scale feeding trial with L. lactis dietary supplement was conducted in a local fish farm (n = 12,000) for three months, and disease resistance, innate immune parameters and growth performance were evaluated. The average weight gain and feed efficiency were increased up to 6.8% and 8.5%, respectively. At the end of the feeding trial, the olive flounders were challenged with S. iniae. The L. lactis-fed group was protected from S. iniae challenge with a 66% survival rate. This disease protection is due to the flounder's innate immunity activated by the L. lactis administration: increased lysosomal activities and production of IL-12 and IFN-γ. These data clearly indicated that L. lactis BFE920 may be developed as a functional feed additive for protection against diseases, and for enhancement of feed efficiency and weight gain in olive flounder farming.

Target-Centered Drug Repurposing Predictions of Human Angiotensin-Converting Enzyme 2 (ACE2) and Transmembrane Protease Serine Subtype 2 (TMPRSS2) Interacting Approved Drugs for Coronavirus Disease 2019 (COVID-19) Treatment through a Drug-Target Interaction Deep Learning Model.

Previously, our group predicted commercially available Food and Drug Administration (FDA) approved drugs that can inhibit each step of the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using a deep learning-based drug-target interaction model called Molecule Transformer-Drug Target Interaction (MT-DTI). Unfortunately, additional clinically significant treatment options since the approval of remdesivir are scarce. To overcome the current coronavirus disease 2019 (COVID-19) more efficiently, a treatment strategy that controls not only SARS-CoV-2 replication but also the host entry step should be considered. In this study, we used MT-DTI to predict FDA approved drugs that may have strong affinities for the angiotensin-converting enzyme 2 (ACE2) receptor and the transmembrane protease serine 2 (TMPRSS2) which are essential for viral entry to the host cell. Of the 460 drugs with Kd of less than 100 nM for the ACE2 receptor, 17 drugs overlapped with drugs that inhibit the interaction of ACE2 and SARS-CoV-2 spike reported in the NCATS OpenData portal. Among them, enalaprilat, an ACE inhibitor, showed a Kd value of 1.5 nM against the ACE2. Furthermore, three of the top 30 drugs with strong affinity prediction for the TMPRSS2 are anti-hepatitis C virus (HCV) drugs, including ombitasvir, daclatasvir, and paritaprevir. Notably, of the top 30 drugs, AT1R blocker eprosartan and neuropsychiatric drug lisuride showed similar gene expression profiles to potential TMPRSS2 inhibitors. Collectively, we suggest that drugs predicted to have strong inhibitory potencies to ACE2 and TMPRSS2 through the DTI model should be considered as potential drug repurposing candidates for COVID-19.

Predicting commercially available antiviral drugs that may act on the novel coronavirus (SARS-CoV-2) through a drug-target interaction deep learning model.

The infection of a novel coronavirus found in Wuhan of China (SARS-CoV-2) is rapidly spreading, and the incidence rate is increasing worldwide. Due to the lack of effective treatment options for SARS-CoV-2, various strategies are being tested in China, including drug repurposing. In this study, we used our pre-trained deep learning-based drug-target interaction model called Molecule Transformer-Drug Target Interaction (MT-DTI) to identify commercially available drugs that could act on viral proteins of SARS-CoV-2. The result showed that atazanavir, an antiretroviral medication used to treat and prevent the human immunodeficiency virus (HIV), is the best chemical compound, showing an inhibitory potency with Kd of 94.94 nM against the SARS-CoV-2 3C-like proteinase, followed by remdesivir (113.13 nM), efavirenz (199.17 nM), ritonavir (204.05 nM), and dolutegravir (336.91 nM). Interestingly, lopinavir, ritonavir, and darunavir are all designed to target viral proteinases. However, in our prediction, they may also bind to the replication complex components of SARS-CoV-2 with an inhibitory potency with Kd  < 1000 nM. In addition, we also found that several antiviral agents, such as Kaletra (lopinavir/ritonavir), could be used for the treatment of SARS-CoV-2. Overall, we suggest that the list of antiviral drugs identified by the MT-DTI model should be considered, when establishing effective treatment strategies for SARS-CoV-2.

Whole Genome Analysis of Lactobacillus plantarum Strains Isolated From Kimchi and Determination of Probiotic Properties to Treat Mucosal Infections by Candida albicans and Gardnerella vaginalis.

Three Lactobacillus plantarum strains ATG-K2, ATG-K6, and ATG-K8 were isolated from Kimchi, a Korean traditional fermented food, and their probiotic potentials were examined. All three strains were free of antibiotic resistance, hemolysis, and biogenic amine production and therefore assumed to be safe, as supported by whole genome analyses. These strains demonstrated several basic probiotic functions including a wide range of antibacterial activity, bile salt hydrolase activity, hydrogen peroxide production, and heat resistance at 70°C for 60 s. Further studies of antimicrobial activities against Candida albicans and Gardnerella vaginalis revealed growth inhibitory effects from culture supernatants, coaggregation effects, and killing effects of the three probiotic strains, with better efficacy toward C. albicans. In vitro treatment of bacterial lysates of the probiotic strains to the RAW264.7 murine macrophage cell line resulted in innate immunity enhancement via IL-6 and TNF-α production without lipopolysaccharide (LPS) treatment and anti-inflammatory effects via significantly increased production of IL-10 when co-treated with LPS. However, the degree of probiotic effect was different for each strain as the highest TNF-α and the lowest IL-10 production by the RAW264.7 cell were observed in the K8 lysate treated group compared to the K2 and K6 lysate treated groups, which may be related to genomic differences such as chromosome size (K2: 3,034,884 bp, K6: 3,205,672 bp, K8: 3,221,272 bp), plasmid numbers (K2: 3, K6 and K8: 1), or total gene numbers (K2: 3,114, K6: 3,178, K8: 3,186). Although more correlative inspections to connect genomic information and biological functions are needed, genomic analyses of the three strains revealed distinct genomic compositions of each strain. Also, this finding suggests genome level analysis may be required to accurately identify microorganisms. Nevertheless, L. plantarum ATG-K2, ATG-K6, and ATG-K8 demonstrated their potential as probiotics for mucosal health improvement in both microbial and immunological contexts.

Multidisciplinary and Comparative Investigations of Potential Psychobiotic Effects of Lactobacillus Strains Isolated From Newborns and Their Impact on Gut Microbiota and Ileal Transcriptome in a Healthy Murine Model.

Psychobiotics are probiotic microorganisms that may exert positive influence on the psychological status of the host. Studies have revealed immunological and microbiological correlations of gut microbiota and the gut-brain axis, and have investigated psychobiotics based on the findings of the gut-brain axis. Considering their mode of actions, the present study sets anti-inflammatory effect, neurotransmitter modulation, and gut microbiota modulation as three essential criteria to evaluate Lactobacillus casei ATG-F1 (F1), L. reuteri ATG-F3 (F3), and L. reuteri ATG-F4 (F4) isolated from newborns as psychobiotics candidates in a healthy mouse model and compares the results with a non-treated control group and an ampicillin-induced gut dysbiosis (Amp) group as a negative control. The F3 and F4 strains showed anti-inflammatory effects in vitro in RAW264.7 murine macrophages, and the level of anti-inflammatory cytokine interleukin (IL)-10 increased in ileums of mice orally administered with the F4 strain. Serum dopamine level significantly increased only in the F4-treated group as compared with the control group. Serum serotonin level was unaffected in Lactobacillus-treated groups, while a significant decrease in serum serotonin level was observed in the Amp group. Bacteroidetes population increased in fecal samples of the F4-treated group as compared with the control, and Bacteroidales S24-7 and Prevotellaceae population significantly increased at family level in fecal samples from the F4-treated group as compared with the control. In contrast, the Amp group showed an increase in the level of Proteobacteria and a decrease in the level of Bacteroidetes as compared with the control group. Transcriptome analysis revealed a distinctive clustering in ileums from the F4-treated group as compared to other experimental groups. In addition, the circadian rhythm pathway showed maximum enrichment in ileums of Lactobacillus-treated mice, and the F4-treated group showed the highest fold changes in circadian rhythm-related genes (Dbp, Per1, Per2, and Per3). Conclusively, L. reuteri ATG-F4 is suggested as a potential psychobiotics through demonstrations of anti-inflammatory effects, serum dopamine modulation, and gut microbiota modulation in a healthy murine model in the present study. Moreover, we carefully suggest gut circadian rhythm modulation as another important criterion of psychobiotics, which may have an important role in the gut-brain axis.

Lactic Acid Bacteria in Finfish-An Update.

A complex and dynamic community of microorganisms, play important roles within the fish gastrointestinal (GI) tract. Of the bacteria colonizing the GI tract, are lactic acid bacteria (LAB) generally considered as favorable microorganism due to their abilities to stimulating host GI development, digestive function, mucosal tolerance, stimulating immune response, and improved disease resistance. In early finfish studies, were culture-dependent methods used to enumerate bacterial population levels within the GI tract. However, due to limitations by using culture methods, culture-independent techniques have been used during the last decade. These investigations have revealed the presence of Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Streptococcus, Carnobacterium, Weissella, and Pediococcus as indigenous species. Numerous strains of LAB isolated from finfish are able to produce antibacterial substances toward different potential fish pathogenic bacteria as well as human pathogens. LAB are revealed be the most promising bacterial genera as probiotic in aquaculture. During the decade numerous investigations are performed on evaluation of probiotic properties of different genus and species of LAB. Except limited contradictory reports, most of administered strains displayed beneficial effects on both, growth-and reproductive performance, immune responses and disease resistance of finfish. This eventually led to industrial scale up and introduction LAB-based commercial probiotics. Pathogenic LAB belonging to the genera Streptococcus, Enterococcus, Lactobacillus, Carnobacterium, and Lactococcus have been detected from ascites, kidney, liver, heart, and spleen of several finfish species. These pathogenic bacteria will be addressed in present review which includes their impacts on finfish aquaculture, possible routes for treatment. Finfish share many common structures and functions of the immune system with warm-blooded animals, although apparent differences exist. This similarity in the immune system may result in many shared LAB effects between finfish and land animals. LAB-fed fish show an increase in innate immune activities leading to disease resistances: neutrophil activity, lysozyme secretion, phagocytosis, and production of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, and TNF-α). However, some LAB strains preferentially induces IL-10 instead, a potent anti-inflammatory cytokine. These results indicate that LAB may vary in their immunological effects depending on the species and hosts. So far, the immunological studies using LAB have been focused on their effects on innate immunity. However, these studies need to be further extended by investigating their involvement in the modulation of adaptive immunity. The present review paper focuses on recent findings in the field of isolation and detection of LAB, their administration as probiotic in aquaculture and their interaction with fish immune responses. Furthermore, the mode of action of probiotics on finfish are discussed.