Two Novel Bacteriophages Control Multidrug- and Methicillin-Resistant Staphylococcus pseudintermedius Biofilm.

As a primary bacterial pathogen in companion animals, Staphylococcus pseudintermedius has zoonotic potential. This pathogen exhibits multidrug resistance, including resistance to methicillin, and biofilm-forming ability, making it hard to eradicate with antimicrobial agents. One potential alternative is bacteriophage therapy. In this study, we first characterized the antimicrobial resistance profile of S. pseudintermedius from canine samples and isolated two novel bacteriophages, pSp-J and pSp-S, from canine pet parks in South Korea to potentially control S. pseudintermedius. The biological characteristics of phages were assessed, and the phages could infect most of the methicillin-resistant S. pseudintermedius strains. We found that these phages were stable under the typical environment of the body (~37°C, pH 7). We also assessed bacterial lysis kinetics using the two phages and their cocktail, and found that the phages could prevent biofilm formation at low doses and could degrade biofilm at high doses. Taken together, this study demonstrates that bacteriophages pSp-J and pSp-S isolated in this study can be used to potentially treat methicillin-resistant S. pseudintermedius.

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.

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.

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.

Isolation of Chryseobacterium siluri sp. nov., from liver of diseased catfish (Silurus asotus).

Yellow-pigmented, circular bacteria (strain SNU WT7) were isolated from the liver of moribund eastern catfish (Silurus asotus). Our study focused on the taxonomic description of SNU WT7 using phylogenetic, phenotypic, and chemotaxonomic analyses. The 16S rRNA gene sequence of the SNU WT7 strain was highly similar to that of Chryseobacterium haifense H38T (97.29% similarity), followed by Chryseobacterium hominis P2K6T (97.22% similarity), while other species exhibited similarity values of less than 97.0%. The genome of strain SNU WT7 displayed average nucleotide identity and genome-to-genome distance values of 72.35% and 22.0%, respectively, which clearly indicated that the novel species was distant from the other Chryseobacterium species, with its closest relative being C. haifense H38T. Furthermore, the phenotypic characteristics, including acid production from glucose, D-fructose, lactose, and maltose, of strain SNU WT 7 differed from those of C. haifense H38T. The major polar lipid of the strain was phosphatidylethanolamine, and several unidentified aminolipids and lipids were also present. Similar to other Chryseobacterium species, the quinone system was composed mainly of MK-6. The genome of SNU WT7 is 2,690,367 bp with a G + C content of 43.6%. Taken together, our data indicate that the isolate SNU WT7 represents a novel species of the genus Chryseobacterium. Thus, we present the name Chryseobacterium siluri sp. nov. for the novel type strain SNU WT7T (KCTC 72626, JCM 33707).

Identification and Genome Analysis of Vibrio coralliilyticus Causing Mortality of Pacific Oyster (Crassostrea gigas) Larvae.

Vibrio coralliilyticus is known as a coral pathogen that also infects marine bivalve larvae worldwide. It is considered to be one of the major constraints in artificial marine bivalve seed production as it causes mortality. In this study, we first isolated and characterized a high virulent of V. coralliilyticus designated as SNUTY-1 that was the cause of Pacific oyster larvae mortality in Korea. In the pathogenicity test, exposure to 2.14 × 105 CFU/mL for 24 h caused mortality to 88.65 ± 2.4% of the tested healthy Pacific oyster larvae. SNUTY-1 showed anti-microbial resistance to ß-lactams, such as penicillins, cephalosporins, and carbapenems. We sequenced and assembled the complete genome of SNUTY-1 (5,842,676 bp), consisting of two chromosomes (Chr I and Chr II) and two plasmids (pSNUTY1 and pSNUTY2). The COG functional analysis confirmed that Chr I had more genes associated with basic cellular functions in comparison to Chr II. The results of the phylogenetic trees based on OrthoANI values indicated that the SNUTY-1 was closely related to V. coralliilyticus strains. SNUTY-1 had a unique plasmid (pSNUTY2), which could mean that the Korean isolate is different from other sequenced V. coralliilyticus strains from different geographical origins. Toxic proteins such as cytolysin/hemolysin and extracellular metalloprotease genes were encoded on Chr I and Chr II of SNUTY-1. These data facilitate the control of V. coralliilyticus infections in aquaculture by providing valuable insights into the biodiversity of this organism and valuable information for the study of virulence factors.

Characterization and Pathological Analysis of a Virulent Edwardsiella anguillarum Strain Isolated From Nile Tilapia (Oreochromis niloticus) in Korea.

Edwardsiella species are one of the top causative pathogens of mortality in various fisheries worldwide. Their role in zoonotic infections and increase in antibiotic-resistance has raised concerns and interests in many research fields. Similar to the studies investigating human clinical cases, there has been an increase in research examining the potential pathogenic role of the bacterium in aquaculture. Within the Edwardsiella family, Edwardsiella anguillarum was lastest group to be differentiated from the Edwardsiella tarda group, and many studies focusing on the virulence of this species have since ensued. In Korea, only E. tarda infections have been reported in aquaculture industries, and there have been no reports on economic losses incurred owing to E. anguillarum infection. There has been a recent report investigating the pathogenicity and pathological changes caused by E. anguillarum infection in a tilapia farm located in the Costa Rica. To the best of our knowledge, as ours is the first report of E. anguillarum infection in a Nile tilapia (Oreochromis niloticus) farm located in an Asian country, the pathogenicity of the bacterial strain was histopathologically compared to that of the past studies. As tilapia is one of the most globally consumed fish species, particularly throughout Asia, Europe, and America, an epidemiological study regarding the disease distribution is necessary for the control and prevention of the disease. Here, we report the first mass mortality case caused by E. anguillarum infection in a Nile tilapia farm located in Korea; the bacterial strain responsible was isolated, characterized, and pathologically analyzed.

Vaccination of fish against Aeromonas hydrophila infections using the novel approach of transcutaneous immunization with dissolving microneedle patches in aquaculture.

The aim of this study was to develop and evaluate a novel route of administration for vaccinating fish against Aeromonas hydrophila infection using a dissolving microneedles (MNs) patch. The A. hydrophila JUNAH strain was inactivated with formalin and used as a vaccine antigen. It was mixed with dissolvable carboxymethyl cellulose (CMC) as the matrix material to produce the MNs patches. When examined with a scanning electron microscope, each patch has 282 uniformly distributed, pyramid-shaped needles on a circular base. In the skin insertion experiment, the MNs patches were confirmed to be capable of penetrating the skin of the fish. Through agglutination assay and analysis of non-specific parameters like lysozyme and superoxide dismutase, it was verified that the antigen embedded into the patch induced adaptive and innate immune responses in the fish. In the challenge experiment, the group inoculated with the MNs patch and the group injected with formalin killed cells (FKC) showed a similar survival rate. Our results suggest that the FKC-loaded MNs patch is a wholly viable method alternative to injection for the vaccination of fish.

Genomic characterization of bacteriophage pEt-SU, a novel phiKZ-related virus infecting Edwardsiella tarda.

A bacteriophage infecting Edwardsiella tarda (named pEt-SU) was isolated from freshwater collected in Chung-ju, South Korea. The whole genome of pEt-SU was 276,734 bp in length, representing the first giant phage infecting Edwardsiella reported to date. A total of 284 putative open reading frames were predicted and annotated. Morphology and genome analyses verified that pEt-SU may be distantly related to the phiKZ-like phages, a well-known giant myovirus. The findings in this study provide new insights into the phages infecting E. tarda ads well as fundamental data for the study of giant phages.

Genome and Phylogenetic Analysis of Infectious Hematopoietic Necrosis Virus Strain SNU1 Isolated in Korea.

Infectious hematopoietic necrosis virus (IHNV), one of the most important pathogenic fish viruses, affects trout fisheries and causes considerable economic losses. Currently, in Korea, more studies on IHNV infection are being reported. However, relatively less data is available on Korean isolates than on those from other countries. Few studies have focused on gene sequence analyses of IHNV glycoprotein (G) gene and almost none have focused on other gene fragments. Therefore, considering the dearth of adequate phylogenetic and genomic studies on Korean IHNV strains because of the lack of data, our study aimed to provide sufficient relevant data by sequencing the complete genome of the IHNV strain SNU1, which was recently isolated from a Korean rainbow trout farm. Moreover, we focused on expanding the perspectives on the phylogenesis of IHNV isolates from Korea and other Asian countries. IHNV was isolated from pooled hematopoietic tissue samples using Epithelioma papulosum cyprinid (EPC) cells, and phylogenetic analysis and genome study were conducted using complete G, N, and nonvirion (NV) gene sequences. Our main achievements were the development of a phylogenetic analytical method based on the NV gene and complete genome sequence analysis of the IHNV strain SNU1, which was compared with other Asian isolate sequences.

Genetic Characterization and Pathological Analysis of a Novel Bacterial Pathogen, Pseudomonas tructae, in Rainbow Trout (Oncorhynchus mykiss).

Pseudomonas species are one of the most prevalent bacterial species globally distributed in forest soil, river water, and human or animal skin. Some species are pathogens or opportunistic pathogens in hospitalized patients, animals, and plants. Various Pseudomonas species, including Pseudomonas putida, P. plecoglossicida, P. aeruginosa, and P. fluorescens, are known fish pathogens; P. fluorescens and P. putida cause severe losses in rainbow trout farming. Therefore, we investigated and isolated the pathogen that is responsible for mortality in a rainbow trout farm in Korea. The isolated bacterium was a strain of P. tructae, which was recently classified in the P. putida group. We performed taxonomical analysis of the bacteria in our previous study. In this study, we investigated the pathogenicity and clinical symptoms of P. tructae and analyzed its genomic characteristics. The pathogenicity of the strain was tested via challenge experiments in healthy rainbow trout and histopathologic analysis of the infected fish. Genome sequence was analyzed to identify the bacterial genes that are involved in antibiotic resistance and virulence. This is the first study reporting P. tructae as an emerging pathogen that is responsible for mortality in rainbow trout fisheries and providing the genome sequence of P. tructae.

Effect of the Algicide Thiazolidinedione 49 on Immune Responses of Bay Scallop Argopecten Irradians.

The thiazolidinedione 49 (TD49) is an effective algaecide against harmful algae; however, its potential effects on the immune function of the edible bay scallop are unclear. Therefore, the present work studied the effects of TD49 on the immune response in bay scallop by evaluating activities of acid phosphatase (ACP), alkaline phosphatase (ALP), and superoxide dismutase (SOD), as well as nitric oxide (NO) levels, total protein content, and expression of immune genes (CTL-6, PGRP, PrxV, MT, and Cu/Zn-SOD) at 3-48 h post-exposure (hpe) to TD49. The activities of ACP and ALP significantly increased in TD49-treated groups at 3-24 hpe, whereas NO levels decreased significantly in 0.58 and 0.68 µM of TD49 at 6-24 hpe, after which the level was similar to that in the untreated control. Moreover, SOD activity significantly increased in all three concentration groups at 3-6 hpe, while it decreased at 12 hpe in the 0.68 µM TD49 treatment group. Notably, total protein content increased with TD49 treatment at each time interval. The results revealed that variable effects on the expression of immune-related genes were observed after treatment with TD49. The findings demonstrate that exposure of scallops to TD49 changes immune responses and expression of immune-related genes. We hypothesize that TD49 may disrupt immune system in bay scallop. The current investigation highlights the potential negative effects of using TD49 as an algaecide on marine economic bivalves to control harmful algal blooms in marine environments.

Janthinobacterium lividum as An Emerging Pathogenic Bacterium Affecting Rainbow Trout (Oncorhynchus mykiss) Fisheries in Korea.

Janthinobacterium spp. are normally considered non-pathogenic, and few pathogenesis-related studies have been reported. Here, we report the first isolation of Janthinobacterium lividum in Korea as a pathogenic bacterium infecting rainbow trout. Mass mortality was observed at one rainbow trout hatchery, and dead fish were necropsied. Gram-negative, nonmotile, rod-shaped bacteria that grew on Cytophaga agar were isolated. A specific violet pigmentation was observed after 7 days of cultivation, and the species were characterized on the basis of the analysis of the 16S rRNA gene. Because no research has focused so far on the pathogenicity of these bacteria, our study was directed to their pathogenic role based on infection-induced histopathology. Examination of stained tissue sections revealed severe renal bacteraemia and tubule degeneration. Other tissue sections, including sections from the liver and the spleen, were relatively clear. The measured half-maximal lethal dose (LD50) was approximately 3 × 105 colony-forming units/fish, suggesting that this bacterium may be an opportunistic pathogen in rainbow trout fisheries. Since the bacterium commonly dwells in soil and most water for rainbow trout fisheries in Korea is supplied from ground water, the bacteria may naturally flow into the aquatic environment. Therefore, recognition of any pathogenic role of J. lividum is important for the prevention of disease in aquaculture.

Application of the bacteriophage pVco-14 to prevent Vibrio coralliilyticus infection in Pacific oyster (Crassostrea gigas) larvae.

Vibrio coralliilyticus infects a variety of shellfish larvae, including Pacific oyster (Crassostrea gigas) larvae worldwide, and remains a major constraint in marine bivalve aquaculture practice, especially in artificial seed production facilities. In this study, we isolated and characterized the bacteriophage (phage) that specifically infects V. coralliilyticus. The phage was designated pVco-14 and classified as Siphoviridae. We also investigated the potential efficacy of the isolated phage against V. coralliilyticus infection. We conducted a survey to replace the overuse of antibiotics, which generate multi-antibiotic-resistant strains and causes environmental pollution. The latent period of pVco-14 was estimated to be approximately 30 min, whereas the burst size was 13.3 PFU/cell. The phage was found to infect four strains of tested V. coralliilyticus. pVco-14 was stable at wide temperature (4-37 °C) and pH (5.0-9.0) ranges. Eighty-one percent of oyster larvae died in an immersion challenge at a dose 1.32 × 105 CFU/ml of virulent V. coralliilyticus (strain 58) within 24 h. When oyster larvae were pre-treated with the phage before the bacterial challenge (bacterial conc.: 1.32 × 104 and 1.32 × 105 CFU/ml), mortality of the phage-treated oyster larvae was lower than that of the untreated control. These results suggest that pVco-14 has potential to be used as a prophylactic agent for preventing V. coralliilyticus infection in marine bivalve hatcheries and can reduce the overuse of antibiotics.

Pseudomonas tructae sp. nov., novel species isolated from rainbow trout kidney.

This study describes the biochemical and phylogenetic characteristics of a Gram-negative strain, SNU WT1T, isolated from rainbow trout kidney. The 16S rRNA gene sequencing indicated that strain SNU WT1T was highly similar to Pseudomonas wadenswilerensis CCOS 864T and closely related to other Pseudomonas putida-related strains. Multilocus sequence analysis of concatenated partial gyrB, rpoB and rpoD sequences revealed that strain SNU WT1T was distinct from P. putida-related strains and formed a separate clade. The average nucleotide identity and Genome-to-Genome Distance Calculator values were 90.19 and 41.7 %with its closest relative P. wadenswilerensis CCOS 864T; however, it was phenotypically distinct from CCOS 864T with respect to arginine dihydrolase, glucose fermentation, aesculin hydrolysis and N-acetyl-glucosamine assimilation. The major polar lipid of the strain was phosphatidylethanolamine and the major quinone was Q-9. The genome of strain SNU WT1T had 5 685 196 bp with a G+C content of 61.83 mol%. We describe a novel species of genus Pseudomonas, for which the name Pseudomonastructae has been proposed, with SNU WT1T (=KCTC 72265=JCM 33436) as the type strain.

Emergence of Rickettsial Infection in Rainbow Trout (Oncorhynchus mykiss) Fry Displaying the Appearance of Red Mark Syndrome in Korea.

Red mark syndrome (RMS) is a fish disease caused by the infection of Rickettsial agents, especially affecting rainbow trout (Oncorhynchus mykiss). The disease is prevalent in many countries in Europe (France, Switzerland, Italy, and Slovenia), South America (Chile), North America (USA), and even Asia (Japan). However, it has not been reported in Korean aquaculture. In February 2019, rainbow trout presenting red spot lesions with swollen features on the lateral side of their body were observed at a hatchery in Korea. Fishes showing those clinical signs were fry weighing 25 ± 5 g. Moreover, the fish showing the red spot lesions were found dead, which suggests an outbreak of a mortality-causing disease. The symptoms were similar to those of RMS, and we identified the presence of Rickettsia-like organisms associated with this disease using polymerase chain reaction (PCR), sequencing, histopathologic examination, and transmission electron microscopy. The distinct features of this infection, compared to that in previous reports, were that RMS occurred in small-sized fish and accompanied mortality. Additionally, the presence of the Rickettsia agent was accompanied with outbreak of the disease. Therefore, this is the first report of RMS outbreak in rainbow trout fisheries in Korea.

Staphylococcus xylosus Infection in Rainbow Trout (Oncorhynchus mykiss) As a Primary Pathogenic Cause of Eye Protrusion and Mortality.

Staphylococcal infections are extensively investigated in humans owing to the resistance of staphylococci to diverse antibiotics commonly used in hospitals. The resistance mechanism of methicillin-resistant Staphylococcus aureus has garnered the interest of researchers due to its risk to the global public health. Furthermore, the zoonotic potential of staphylococci has led to increased interest in their transmission mechanism via food, livestock, as well as domestic and wild animals. Although fish are globally consumed, there are only few studies on the potential threat of staphylococcal infection in aquatic animals. In this study, we present the first description of Staphylococcus xylosus infection and its pathogenicity in rainbow trout, which resulted in fish mortality and economic losses in trout fisheries. We focused on the pathogenic role of the bacterium and its influence on rainbow trout based on the clinical symptoms in the eyes. Staphylococcus xylosus infection induced exophthalmia and disrupted the primary immune barrier, which increased the possibility of other secondary bacterial infections in fish under poor conditions, resulting in continuous mortality.

Detoxification, Apoptosis, and Immune Transcriptomic Responses of the Gill Tissue of Bay Scallop Following Exposure to the Algicide Thiazolidinedione 49.

Thiazolidinedione 49 (TD49), a newly synthesized algicide, shows strong toxicity at low concentrations of 0.1-2.0 µM. However, its potential effects on non-target species at the transcript level were not investigated. Differentially expressed genes (DEGs) in the gills of the bay scallop, Argopecten irradians, were accessed after treatment with 0.68 µM TD49 for up to 48 h. Following exposure, it was observed that 5214 genes were upregulated and 3497 were downregulated. Functional enrichment analysis revealed that the apoptosis pathway was activated. The extrinsic apoptosis pathway was activated and the survival factors related pathway was suppressed. Furthermore, gene expressions related to ATP-binding cassette, nuclear factor erythroid 2-related factor, B cell lymphoma-2 family protein, glutathione reductase, glutathione peroxidase, catalase, NADPH2:quinone reductase, and superoxide dismutase were decreased. Conversely, gene expressions related to FAS-associated death domain protein, glutathione S-transferase, caspase 6, 8, cytochrome P450 1A1, and 2C8 were increased. These results comprehensively demonstrated the toxicity of the novel algicide TD49, and should draw the attention of researchers to the importance of analyzing the potential impact of chemical compounds as algicides to control the proliferation of harmful algae, due to the secondary pollution caused by their application.

Enhanced bath immersion vaccination through microbubble treatment in the cyprinid loach.

Immunization by bath immersion is likely the simplest method of fish vaccination. Although the route of immunogenicity has not been fully identified, immersion vaccination is clearly a useful labor-saving technique. In this study, microbubble (MB) treatment was assessed for its ability to improve the efficacy of bath immersion vaccination in the cyprinid loach. MBs are commonly defined as minute particles of gas with a diameter of less than 100 µm, which generated free radicals. Here, the efficacy of MB treatment for vaccination enhancement in the cyprinid loach was assessed in direct challenge experiments using the virulent Aeromonas hydrophila JUNAH strain; assessments comprised agglutination titer assay and non-specific parameter analysis. Agglutination titers were high in loaches that were immunized via injection with inactivated cells (FKC group); however, non-specific immune activation parameters (e.g., lysozyme, superoxide dismutase, and phagocytic activity) were more increased in loaches that were immunized via bath immersion with MB treatment. Moreover, MB-treated loaches showed comparable survival rates, relative to loaches immunized via injection with formalin inactivated cells. Thus, higher levels of non-specific immune parameters suggest increased efficacy of this vaccine approach. Improving the effectiveness of bath immersion vaccine will increase its affordability and ease of application in aquaculture.

Isolation and characterisation of pVa-21, a giant bacteriophage with anti-biofilm potential against Vibrio alginolyticus.

There is an increasing emergence of antibiotic-resistant Vibrio alginolyticus, a zoonotic pathogen that causes mass mortality in aquatic animals and infects humans; therefore, there is a demand for alternatives to antibiotics for the treatment and prevention of infections caused by this pathogen. One possibility is through the exploitation of bacteriophages. In the present study, the novel bacteriophage pVa-21 was classified as Myoviridae and characterised as a candidate biocontrol agent against V. alginolyticus. Its morphology, host range and infectivity, growth characteristics, planktonic or biofilm lytic activity, stability under various conditions, and genome were investigated. Its latent period and burst size were estimated to be approximately 70 min and 58 plaque-forming units/cell, respectively. In addition, phage pVa-21 can inhibit bacterial growth in both the planktonic and biofilm states. Furthermore, phylogenetic and genome analysis revealed that the phage is closely related to the giant phiKZ-like phages and can be classified as a new member of the phiKZ-like bacteriophages that infect bacteria belonging to the family Vibrionaceae.