Probiotics in addressing heavy metal toxicities in fish farming: Current progress and perspective.

Heavy metal contamination of aquatic environments adversely affects the health of aquatic organisms and consumption of fish contaminated with heavy metals poses serious health risks to humans. Among various strategies, probiotics (living microorganisms known to have beneficial effects on the host), which have been extensively applied in the aquaculture industry, could be helpful for heavy metal detoxification and remediation. Several probiotics, including Lactobacillus strains, exhibit heavy metal binding, high heavy metal tolerance, and other beneficial characteristics for the host. Notably, numerous probiotics have been reported to bind heavy metals and excrete them from the host. Various probiotic strains (Lactobacillus, Bacillus, Lactococcus, etc.) show beneficial effects in alleviating heavy metal toxicity in cultured fish species. Certain probiotic bacteria reduce the absorption and bioavailability of heavy metals by enhancing heavy metal detoxification and sequestration while preserving gut barrier function. This review summarises the toxic effects of selected heavy metals on the health of farmed fish and discusses the role of probiotic strains in remediating the consequential exposure-induced immune toxicity and oxidative stress. Moreover, we discussed the protective strategies of probiotics against heavy metal accumulation in various tissues and gut dysbiosis in fish to alleviate heavy metal toxicity in fish farming, thereby promoting a sustainable blue economy worldwide.

First report of Perkinsus marinus occurrence associated with wild Pacific oysters Crassostrea gigas from the west coast of Korea.

This study reports the occurrence of Perkinsus marinus associated with wild Pacific oyster (Crassostrea gigas) specimens collected along the west coast of Korea. Confirmation of P. marinus presence was achieved by conventional PCR using World Organization of Animal Health (WOAH)-recommended primers that specifically targeted regions of the rDNA locus (ITS1, 5.8S, and ITS2). Sequencing of 10 samples revealed two distinct sequences differing by a single base pair, indicating potential haplotype variability. One sequence closely resembled the P. marinus strain found in Maryland, USA, whereas the other exhibited divergence, indicative of species diversity in the Korean strain, as was evident from the haplotype network analysis. Further validation involved the Ray's Fluid Thioglycollate Medium (RFTM) assay, which initially yielded inconclusive results, possibly due to low infection intensity. Subsequently, RFTM and 2 M NaOH assays conducted on the isolates in the present study, cultured P. marinus cells in standard DMEM/F12 medium, and a positive P. marinus strain (ATCC 50509), revealed characteristic hypnospores of P. marinus upon Lugol's iodine staining. These comprehensive investigations underscore the conclusive confirmation of P. marinus in Korean waters and mark a significant milestone in our understanding of the distribution and characteristics of this parasite in previously unreported regions.

Impact of dietary piperine on growth performance, immune response, antioxidant status, and immune-related gene expression in pathogen-aggravated Cyprinus carpio.

This study investigated the effects of dietary piperine (PIP) on growth performance, digestive enzymes, serum biochemical parameters, antioxidant and immune responses, and gene expression in Cyprinus carpio challenged with Aeromonas hydrophila. Six diets were prepared with PIP doses of 0, 0.5, 1.0, 2.0, 3.0, and 4.0 g/kg, corresponding with the control, PR50, PR100, PR200, PR300, and PR400, respectively. Fish were challenged with Aeromonas hydrophila after 8 weeks of feeding with the respective diets. Weight gain (PWG) and specific growth rate (SGR) were significantly enhanced, whereas feed conversion ratio (FCR) was lowered in PR200. The cumulative post-challenge survival was improved to 68.43% in the PR200 group compared with 28.08% in the control. Serum total protein and albumin levels were significantly enhanced in the PR200 group compared to the control. However, dietary PIP up to 3 g/kg had no significant effect on serum glucose, cortisol, aspartate aminotransferase, or alkaline phosphatase activities; however, the alanine aminotransferase level was lower (P < 0.05) in the PR200 group than in the control. Intestinal amylase, lipase, and protease activities increased in PR300, and intestinal amylase and lipase increased in the PR100 group (P < 0.05). The serum immunological indices (lysozyme, alternative complement pathway, phagocytic activity, and respiratory burst activity) were higher (P < 0.05) in the PR200 group than in the control group. Serum superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities were significantly intensified in PR200-PR300 than in the control group, with the highest activity observed in the PR200 group. Malondialdehyde was significantly lower in the PR200 group than in the control group. Furthermore, SOD, CAT, and Nrf2 expression was strongly upregulated in the liver tissue of the PR200 and PR300 groups compared to that in the control. The transcript levels of pro-inflammatory cytokines viz. IL-1ß and TNF-α were significantly upregulated in the kidneys of the PR100 and PR200 post-challenged. In contrast, the anti-inflammatory cytokine IL-10 was significantly downregulated in the kidneys of PR200. The expression of HSP70 was upregulated only in the PR400. Quadratic regression analysis showed that the optimal dietary PIP level was estimated as 2.07-2.13 g/kg to maximize growth performance. Overall, these results indicate that dietary PIP at an appropriate level can improve immunity, cytokine gene expression, and disease resistance in C. carpio.

Bactericidal efficacy of non-thermal plasma activation against Aeromonas hydrophila and immunological responses of koi (Cyprinus carpio haematopterus).

In the aquaculture industry, an efficient and safe water purification system is important to prevent mass mortality by virulent pathogens. As extensive use of traditional methods (e.g.: povidone-iodine, ozone, ultraviolet irradiation, formalin, and chlorine dioxide) have adverse effects on cultured fish, an appropriate and alternative water purification method is vital for the sustainability of the industry. Non-thermal plasma technology has been successfully used for various biomedical purposes (e.g: food sterilization, medical device disinfection, wound healing, cancer therapy, etc.) and has great potential to be used as a sterilizing system. However, few studies have been conducted on its usefulness in the aquaculture industry. In this study, we investigated the bactericidal efficacy of plasma-activated water induced by non-thermal plasma and its histopathological as well as immunological adverse effects on koi. A highly virulent Aeromonas hydrophila SNU HS7, which caused massive mortality of koi, was used for this study. Non-thermal plasma was applied for 10 min to the fish tanks with 1.2 × 109 CFU/mL SNU HS7 using PLMB-20 system to confirm the sterilization efficacy and to observe the survival and immunological reaction of koi for 14 days. As a result, gross pathological, histopathological, and immunological investigations did not reveal any significant adverse effects in fish as compared to the control groups. To the best of our knowledge, this is the first study showing that non-thermal plasma can be used for sterilization of rearing water without giving significant physiological damage to the fish, even under the assumption of extreme situations. As plasma can effectively sterilize not only bacteria but also other unknown pathogens, the results of this study are showing a promising future in purifying water in aquaculture practice.

Genomic characterization of bacteriophage pSal-SNUABM-01, a novel elongated-head phage infecting Salmonella sp.

Salmonellosis is a disease of critical concern for public health, and the use of bacteriophages is among the most promising approaches to combating Salmonella. As Salmonella has various serotypes and strains, and bacteriophages are virulent to specific hosts, it is important to isolate phages and evaluate interactions with their hosts. In the present study, a novel Salmonella-infecting bacteriophage, pSal-SNUABM-01, was isolated and characterized. Transmission electron microscopy revealed that the bacteriophage is a member of the family Podoviridae and possesses an elongated head and a short tail. The phage genome is circular and 89,500 bp in size. A total of 162 open reading frames were predicted, eight of which were tRNAs. Morphological and genomic analysis revealed that pSal-SNUABM-01 is closely related to phage 7-11. In phylogenetic analysis, pSal-SNUABM-01 and 7-11 did not cluster together with the members of any established genus, suggesting that these two phages comprise a novel genus. The results of this study enhance our understanding of the phylogeny of the family Podoviridae and might be applicable to the development of bacteriophage treatments against Salmonella infections.

A Case of Submandibular Leiomyosarcoma, Mimicking an Abscess, in a Ball Python (Python regius).

A two-year-old ball python with a submandibular mass was evaluated. Fine needle aspiration resulted in debris containing purulent materials and bacterial cells on cytology. Radiography demonstrated multi-focal radiopaque lesions in the mass, which were suspected to be mineralization; there was an absence of mandibular invasion or lung involvement. Gross examination of the surgically excised mass revealed a multi-nodular, well-circumscribed lesion with purulent material. The postoperative recovery was uneventful. The histopathological examination followed by immunohistochemistry analysis gave a diagnosis of leiomyosarcoma. As tumors containing purulent materials can be confused with an abscess, diagnostic confirmation with various diagnostical tools should be considered.

Effects of Dietary Lactiplantibacillus plantarum subsp. plantarum L7, Alone or in Combination with Limosilactobacillus reuteri P16, on Growth, Mucosal Immune Responses, and Disease Resistance of Cyprinus carpio.

Skin mucosal lymphoid tissues of fish are the first line of defence against pathogen invasion. We investigated the effects of Lactiplantibacillus plantarum subsp. plantarum L7, singularly or in combination with Limosilactobacillus reuteri P16, on mucosal immunity and diseases resistance of carp Cyprinus carpio. C. carpio (average weight: 26.28 ± 1.02 g) were divided into five experimental groups. Fish in each group were fed with one of the following potential probiotic-supplemented diets: control (0 - basal diet), D1 (107 CFU/g L7), D2 (108 CFU/g L7), D3 (109 CFU/g L7), and D4 (108 CFU/g L7 + 108 CFU/g P16). Eight weeks post-feeding, growth performance was higher in D4, with a final weight gain of 67.18 ± 1.47 g. Results showed a significantly higher skin mucosal lysozyme, alkaline phosphatase, mucus protein level, superoxide dismutase, and catalase activities in D2 and D4 compared to the control. However, potential probiotics had no significant effect on skin mucosal immunoglobulin level. Skin mucus of D4 exhibited stronger inhibition zones against pathogenic bacterial strains. Moreover, digestive enzyme activities (protease, lipase) were highest in D4. Intesinal lactic acid bacterial counts of fish fed combind probiotics (i.e. D4) was significantly higher than the control. Further, supplementation of potential probiotics altered the expression of IL-1ß, TNF-α, and IL-10 cytokines. Fish from D4 exhibited significantly higher relative post-challenge survival (69.7%) against Aeromonas hydrophila, followed by D2 (66.67%). Therefore, the inclusion of L. plantarum subsp. plantarum L7 at 108 CFU/g or in combination with L. reuteri P16 could enhance the growth performance, mucosal immune responses, and disease resistance of C. carpio.

Janthinobacterium tructae sp. nov., Isolated from Kidney of Rainbow Trout (Oncorhynchus mykiss).

This study presents a novel Janthinobacterium strain, SNU WT3, isolated from the kidney of rainbow trout. A phylogenetic study using 16S rRNA sequences indicated that the strain is closely related to Janthinobacterium svalbardensis JA-1T. However, biochemical analysis found differences in D-xylose adonitol, N-acetylglucosamine, arbutin, and cellobiose. As for genome-to-genome distance and average nucleotide identity values calculated between strain SNU WT3 and other related strains such as J. lividum EIF1, J. svalbardensis PAMC 27463, and J. agaricidamnosum BHSEK were all below the cutoff value between species. DNA-DNA hybridization between strain SNU WT3 and other close relatives indicated the results of J. lividum DSM 1522T (47.11%) and J. svalbardensis JA-1T (38.88%) individually. The major fatty acid compositions of strain SNU WT3 were cylco-C17:0 (41.45%), C16:0 (33.86%) and C12:0 (5.87%). The major polar lipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, and diphosphatidylglycerol. The quinone system was composed mainly of ubiquinone Q-8. The genome of strain SNU WT3 consists of 6,314,370 bp with a G + C content of 62.35%. Here, we describe a novel species of the genus Janthinobacterium, and the name Janthinobacterium tructae has been proposed with SNU WT3T (=KCTC 72518 = JCM 33613) as the type strain.

Strategy for mass production of lytic Staphylococcus aureus bacteriophage pSa-3: contribution of multiplicity of infection and response surface methodology.

BACKGROUND: Antibiotic-resistant bacteria have emerged as a serious problem; bacteriophages have, therefore, been proposed as a therapeutic alternative to antibiotics. Several authorities, such as pharmacopeia, FDA, have confirmed their safety, and some bacteriophages are commercially available worldwide. The demand for bacteriophages is expected to increase exponentially in the future; hence, there is an urgent need to mass-produce bacteriophages economically. Unlike the replication of non-lytic bacteriophages, lytic bacteriophages are replicated by lysing host bacteria, which leads to the termination of phage production; hence, strategies that can prolong the lysis of host bacteria in bacteria-bacteriophage co-cultures, are required. RESULTS: In the current study, we manipulated the inoculum concentrations of Staphylococcus aureus and phage pSa-3 (multiplicity of infection, MOI), and their energy sources to delay the bactericidal effect while optimizing phage production. We examined an increasing range of bacterial inoculum concentration (2 × 108 to 2 × 109 CFU/mL) to decrease the lag phase, in combination with a decreasing range of phage inoculum (from MOI 0.01 to 0.00000001) to delay the lysis of the host. Bacterial concentration of 2 × 108 CFU/mL and phage MOI of 0.0001 showed the maximum final phage production rate (1.68 × 1010 plaque forming unit (PFU)/mL). With this combination of phage-bacteria inoculum, we selected glycerol, glycine, and calcium as carbon, nitrogen, and divalent ion sources, respectively, for phage production. After optimization using response surface methodology, the final concentration of the lytic Staphylococcus phage was 8.63 × 1010 ± 9.71 × 109 PFU/mL (5.13-fold increase). CONCLUSIONS: Therefore, Staphylococcus phage pSa-3 production can be maximized by increasing the bacterial inoculum and reducing the seeding phage MOI, and this combinatorial strategy could decrease the phage production time. Further, we suggest that response surface methodology has the potential for optimizing the mass production of lytic bacteriophages.

Citrobacter tructae sp. nov. Isolated from Kidney of Diseased Rainbow Trout (Oncorhynchus mykiss).

A novel Citrobacter species was isolated from the kidney of diseased rainbow trout (Oncorhynchus mykiss) reared on a trout farm. Biochemical characterization and phylogenetic analysis were performed for bacterial identification. Sequencing of the 16S rRNA gene and five housekeeping genes indicated that the strain belongs to the Citrobacter genus. However, multilocus sequence analysis, a comparison of average nucleotide identity, and genome-to-genome distance values revealed that strain SNU WT2 is distinct and forms a separate clade from other Citrobacter species. Additionally, the phenotype characteristics of the strain differed from those of other Citrobacter species. Quinone analysis indicated that the predominant isoprenoid quinone is Q-10. Furthermore, strain virulence was determined by a rainbow trout challenge trial, and the strain showed resistance to diverse antibiotics including ß-lactams, quinolone, and aminoglycosides. The complete genome of strain SNU WT2 is 4,840,504 bp with a DNA G + C content of 51.94% and 106,068-bp plasmid. Genome analysis revealed that the strain carries virulence factors on its chromosome and antibiotic resistance genes on its plasmid. This strain represents a novel species in the genus Citrobacter for which the name C. tructae has been proposed, with SNU WT2 (=KCTC 72517 = JCM 33612) as the type strain.

Bacteriophage as an alternative to prevent reptile-associated Salmonella transmission.

Salmonellosis is a major global public health issue; its most common infection, gastroenteritis, accounts for approximately 90 million illnesses and 150,000 mortalities per year. Eradicating salmonellosis requires surveillance, prevention and treatment, entailing large expenditures. However, it is difficult to control Salmonella transmission because it occurs via multiple routes; exotic reptiles are a reservoir of Salmonella and comprise one such route. As the popularity of exotic pets and animal exhibition has increased, human encounters with reptiles have also increased. As a result, reptile-associated salmonellosis (RAS) has been recognized as an emerging disease. The development of antimicrobial resistance in RAS-causing Salmonella sp. requires alternatives to antibiotics. In this study, bacteriophages have been established as an alternative to antibiotics because only target bacteria are lysed; thus, they are promising biocontrol agents. Here, bacteriophage pSal-SNUABM-02, which infects and lyses reptile Salmonella isolates, was isolated and characterized. The morphology, host range, growth traits and stability of the phage were investigated. The phage was assigned to Myoviridae and was stable in the following conditions: pH 5-9, 4-37°C, and ultravioletA/ultravioletB (UVA/UVB) exposure. Salmonella clearance efficacy was tested using planktonic cell lysis activity and biofilm degradation on polystyrene 96-well plates and reptile skin fragments. The phage exhibited vigorous lysis activity against planktonic cells. In in vitro biofilm degradation tests on reptile skin and polystyrene plates, both low- and high-concentration phage treatments lowered bacterial cell viability by approximately 2.5-3 log colony-forming units and also decreased biomass. Thus, bacteriophages are a promising alternative to antibiotics for the prevention and eradication of RAS.

Cross-sectional anatomy, computed tomography, and magnetic resonance imaging of the banded houndshark (Triakis scyllium).

Due to their important phylogenetic position among extant vertebrates, sharks are an invaluable group in evolutionary developmental biology studies. A thorough understanding of shark anatomy is essential to facilitate these studies and documentation of this iconic taxon. With the increasing availability of cross-sectional imaging techniques, the complicated anatomy of both cartilaginous and soft tissues can be analyzed non-invasively, quickly, and accurately. The aim of this study is to provide a detailed anatomical description of the normal banded houndshark (Triakis scyllium) using computed tomography (CT) and magnetic resonance imaging (MRI) along with cryosection images. Three banded houndsharks were scanned using a 64-detector row spiral CT scanner and a 3 T MRI scanner. All images were digitally stored and assessed using open-source Digital Imaging and Communications in Medicine viewer software in the transverse, sagittal, and dorsal dimensions. The banded houndshark cadavers were then cryosectioned at approximately 1-cm intervals. Corresponding transverse cryosection images were chosen to identify the best anatomical correlations for transverse CT and MRI images. The resulting images provided excellent detail of the major anatomical structures of the banded houndshark. The illustrations in the present study could be considered as a useful reference for interpretation of normal and pathological imaging studies of sharks.

Characterization of Novel Erwinia amylovora Jumbo Bacteriophages from Eneladusvirus Genus.

Jumbo phages, which have a genome size of more than 200 kb, have recently been reported for the first time. However, limited information is available regarding their characteristics because few jumbo phages have been isolated. Therefore, in this study, we aimed to isolate and characterize other jumbo phages. We performed comparative genomic analysis of three Erwinia phages (pEa_SNUABM_12, pEa_SNUABM_47, and pEa_SNUABM_50), each of which had a genome size of approximately 360 kb (32.5% GC content). These phages were predicted to harbor 546, 540, and 540 open reading frames with 32, 34, and 35 tRNAs, respectively. Almost all of the genes in these phages could not be functionally annotated but showed high sequence similarity with genes encoded in Serratia phage BF, a member of Eneladusvirus. The detailed comparative and phylogenetic analyses presented in this study contribute to our understanding of the diversity and evolution of Erwinia phage and the genus Eneladusvirus.

Isolation and Characterization of Salmonella Jumbo-Phage pSal-SNUABM-04.

The increasing emergence of antimicrobial resistance has become a global issue. Therefore, many researchers have attempted to develop alternative antibiotics. One promising alternative is bacteriophage. In this study, we focused on a jumbo-phage infecting Salmonella isolated from exotic pet markets. Using a Salmonella strain isolated from reptiles as a host, we isolated and characterized the novel jumbo-bacteriophage pSal-SNUABM-04. This phage was investigated in terms of its morphology, host infectivity, growth and lysis kinetics, and genome. The phage was classified as Myoviridae based on its morphological traits and showed a comparatively wide host range. The lysis efficacy test showed that the phage can inhibit bacterial growth in the planktonic state. Genetic analysis revealed that the phage possesses a 239,626-base pair genome with 280 putative open reading frames, 76 of which have a predicted function and 195 of which have none. By genome comparison with other jumbo phages, the phage was designated as a novel member of Machinavirus composed of Erwnina phages.

Bacteriophage Cocktail for the Prevention of Multiple-Antibiotic-Resistant and Mono-Phage-Resistant Vibrio coralliilyticus Infection in Pacific Oyster (Crassostrea gigas) Larvae.

Vibrio coralliilyticus (V. coralliilyticus) is a pathogen that causes mass mortality in marine bivalve hatcheries worldwide. In this study, we used a bacteriophage (phage) cocktail to prevent multiple-antibiotic-resistant (MAR) and phage-resistant (PR) V. coralliilyticus infection in Pacific oyster (Crassostrea gigas) larvae. To prevent the occurrence of phage-resistant strains and decrease the effect of mono-phage treatment, we prepared a phage cocktail containing three types of V. coralliilyticus-specific phages and tested its prophylactic efficacy against MAR and PR V. coralliilyticus infection. The results of the cell lysis test showed that the phage cocktail showed an excellent bactericidal effect against the MAR and PR variants in contrast to the experimental group treated with two mono phages (pVco-5 and pVco-7). An in vivo test using Pacific oyster larvae also confirmed the preventive effect against MAR and PR variants. In conclusion, the application of the phage cocktail effectively prevented V. coralliilyticus infection in marine bivalve seedling production. Furthermore, it is expected to reduce damage to the aquaculture industry caused by the occurrence of MAR and PR V. coralliilyticus. Therefore, phage cocktails may be used for the control of various bacterial diseases.

Immunomodulatory Role of Microbial Surfactants, with Special Emphasis on Fish.

Microbial surfactants (biosurfactants) are a broad category of surface-active biomolecules with multifunctional properties. They self-assemble in aqueous solutions and are adsorbed on various interfaces, causing a decrease in surface tension, as well as interfacial tension, solubilization of hydrophobic compounds, and low critical micellization concentrations. Microbial biosurfactants have been investigated and applied in several fields, including bioremediation, biodegradation, food industry, and cosmetics. Biosurfactants also exhibit anti-microbial, anti-biofilm, anti-cancer, anti-inflammatory, wound healing, and immunomodulatory activities. Recently, it has been reported that biosurfactants can increase the immune responses and disease resistance of fish. Among various microbial surfactants, lipopeptides, glycolipids, and phospholipids are predominantly investigated. This review presents the various immunological activities of biosurfactants, mainly glycolipids and lipopeptides. The applications of biosurfactants in aquaculture, as well as their immunomodulatory activities, that make them novel therapeutic candidates have been also discussed in this review.

Effectiveness of the guava leaf extracts against lipopolysaccharide-induced oxidative stress and immune responses in Cyprinus carpio.

The anti-inflammatory activity of the guava leaf extracts (GLE) against LPS-induced inflammatory responses in fish macrophage cell lines is well documented. Here, we evaluated the effects of dietary GLE on LPS-induced oxidative stress, immune responses, and glucocorticoid receptor-related gene expression in Cyprinus carpio. Basal diet was supplemented with 0 (control), 100, 150, 200, or 250 mg kg-1 GLE for eight weeks. Highest (p < 0.05) weight gain rate was obtained in fish group supplemented with 200 mg kg-1 of GLE. The results showed that superoxide dismutase, glutathione-S-transferase, glutathione peroxidase, glutathione reductase, lysozyme, and complement C3 decreased, while malondialdehyde level increased in the liver and spleen upon LPS-challenge. Dietary GLE supplementation (especially 200 or 250 mg kg-1) alleviated LPS-induced changes. Similarly, GLE (150-250 mg kg-1) reversed LPS-induced alteration of serum biochemical parameters such as alkaline phosphatase, aspartate transaminase, alanine transaminase, and myeloperoxidase. LPS treatment markedly induced increased the mRNA levels of TNF-α, IL-1ß, and NF-κB p65 in both the liver and kidney tissues; however, GLE pre-treatment attenuated LPS-induced elicitation of TNF-α, IL-ß, and NF-κB p65. Moreover, dietary GLE supplementation significantly increased the expression of HSP70 and HSP90, and glucocorticoid receptor in the liver and kidney after LPS challenge. Thus, GLE attenuated LPS-induced inflammation response by up-regulating glucocorticoid receptor-related gene expression in carp. Finally, GLE supplementation reduced carp mortality after LPS-challenge. These results suggest that dietary supplementation with 200 mg kg-1 GLE is adequate for effectively attenuating LPS-induced oxidative stress and immune-suppressive effects in C. carpio.

Isolation and Characterization of Two Bacteriophages and Their Preventive Effects against Pathogenic Vibrio coralliilyticus Causing Mortality of Pacific Oyster (Crassostrea gigas) Larvae.

Vibrio coralliilyticus is one of the major pathogens causing mass mortality in marine bivalve larvae aquaculture. To prevent and control Vibrio spp. infections in marine bivalve hatcheries, various antibiotics are overused, resulting in environmental pollution and the creation of multi-drug-resistant strains. Therefore, research on the development of antibiotic substitutes is required. In this study, we isolated two bacteriophages (phages) that specifically infected pathogenic V. coralliilyticus from an oyster hatchery and designated them as pVco-5 and pVco-7. Both phages were classified as Podoviridae and were stable over a wide range of temperatures (4-37 °C) and at pH 7.0-9.0. Thus, both phages were suitable for application under the environmental conditions of an oyster hatchery. The two phages showed confirmed significant bactericidal efficacy against pathogenic V. coralliilyticus in an in vitro test. In the in vivo experiment, the phage pre-treated groups of Pacific oyster larvae showed significantly lower mortality against V. coralliilyticus infection than untreated control larvae. The results of the present study suggest that both phages could be used in the artificial marine bivalve seedling industry; not only to prevent pathogenic V. coralliilyticus infection, but also to reduce antibiotic overuse.

Immunostimulation by starch hydrogel-based oral vaccine using formalin-killed cells against edwardsiellosis in Japanese eel, Anguilla japonica.

Edwardsiellosis outbreaks cause significant losses in Japanese eel aquaculture. The causative agent, Edwardsiella tarda, is an intracellular pathogen, and the use of antibiotics has a limited effectiveness. As Japanese eels are sensitive to stress, injection vaccines are not recommended for treatment; immersion methods are less stressful, but not cost-effective. Alternatively, oral vaccination methods are more promising. The aim of this study was to develop a starch hydrogel-based oral (SHO) vaccine against edwardsiellosis in Japanese eel, using formalin-killed cells. To assess the protective effect, we compared SHO vaccine with the conventional formalin-killed cell (FKC) vaccine. A bacterial agglutination test showed that agglutination titers in SHO-vaccinated group were higher than in the FKC-vaccinated group. Japanese eel survival rate (%) was monitored after challenge by E. tarda at four weeks post-vaccination. Survival rates in the FKC group (60%, first trial; 70%, second trial) were lower than in SHO groups. Percentage survival rates in three SHO groups (first and second trials, respectively) were as follows: 70% and 80% in the group vaccinated once per day for one day; and 80% and 90% in both groups vaccinated for four and eight days. Additionally, a boost SHO vaccination at 46 days prompted a similar or even higher protection against edwardsiellosis than after the initial vaccination. Both FKC and SHO vaccination upregulated levels of pro-inflammatory cytokines (interleukin (IL)-6, tumor necrosis factor (TNF)-α), and host defense cytokine (interferon (IFN)-α) in all immunized groups of fish when compared with the control. These results reveal the immunostimulation effect of SHO vaccine in Japanese eel, emphasizing its potential as an oral vaccine in aquaculture.

Synergistic phage-surfactant combination clears IgE-promoted Staphylococcus aureus aggregation in vitro and enhances the effect in vivo.

Currently, topical antibiotic treatment is a major strategy for decolonisation of Staphylococcus aureus, although it may result in antibiotic resistance or recolonisation of the organism. Recently, application of bacteriophages in the treatment of S. aureus infection has attracted attention. However, a single administration of bacteriophages did not effectively decolonise S. aureus in our first trial in vivo. Using a bacteriophage (pSa-3) and surfactant combination in vitro, we showed an increased (>8%) adsorption rate of the bacteriophage on the host. Moreover, the combination increased the eradication of immunoglobulin E (IgE)-stimulated aggregation, as the surfactant promoted the dissociation of S. aureus aggregates by decreasing the size by 75% and 50% in the absence and presence of IgE, respectively. Furthermore, the combined treatment significantly decolonised the pathogen with an efficacy double that of the phage-only treatment, and decreased the expression of pro-inflammatory cytokine genes (IL-1ß, IL-12 and IFNγ) for 5 days in the second in vivo trial. These results suggest that the bacteriophage-surfactant combination could act as an alternative to antibiotics for S. aureus decolonisation in patients with dermatitis.