Bacteriophage EPP-1, a potential antibiotic alternative for controlling edwardsiellosis caused by Edwardsiella piscicida while mitigating drug-resistant gene dissemination.

Edwardsiella piscicida causes significant economic losses to the aquaculture industry worldwide. Phage-based biocontrol methods are experiencing a renaissance because of the spread of drug-resistant genes and bacteria resulting from the heavy use of antibiotics. Here, we showed that the novel Edwardsiella phage EPP-1 could achieve comparable efficacy to florfenicol using a zebrafish model of Edwardsiella piscicida infection and could reduce the content of the floR resistance gene in zebrafish excreta. Specifically, phage EPP-1 inhibited bacterial growth in vitro and significantly improved the zebrafish survival rate in vivo (P = 0.0035), achieving an efficacy comparable to that of florfenicol (P = 0.2304). Notably, integrating the results of 16S rRNA sequencing, metagenomic sequencing, and qPCR, although the effects of phage EPP-1 converged with those of florfenicol in terms of the community composition and potential function of the zebrafish gut microbiota, it reduced the floR gene content in zebrafish excreta and aquaculture water. Overall, our study highlights the feasibility and safety of phage therapy for edwardsiellosis control, which has profound implications for the development of antibiotic alternatives to address the antibiotic crisis.

Therapeutic efficacies of two newly isolated Edwardsiella phages against Edwardsiella piscicida infection.

The spread of multi-drug resistant (MDR) bacteria has posed a threat to the development of aquaculture. Due to its effective bactericidal ability, phage therapy has been considered as an alternative to antibiotics to reduce infection caused by MDR bacteria. In this study, two Edwardsiella piscicida phages were newly-isolated and characterized to prevent or treat infection in aquaculture. The phages were designated as vB_EpM_ZHS and vB_EpP_ZHX belonging to Myoviridae and Podoviridae families, respectively, in terms of genome sequence and morphology analyses. The combination of vB_EpM_ZHS and vB_EpP_ZHX improved the therapeutic efficacy both in vitro and in vivo. The phage cocktail significantly inhibited bacterial growth in vitro and decreased approximately 40% of mortality rate and an order of magnitude of bacterial burden in zebrafish and turbot infected by E. piscicida. Moreover, the phage cocktail increased transcription levels of tumor necrosis factor-alpha (TNF-α), interleukin-12 (IL-12), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) and alleviated inflammatory levels in the hindgut and spleen of turbots. The results indicate that the phage has a promising potential for therapeutic use against E. piscicida as the antimicrobial alternative to antibiotics in aquaculture.

Phenotypic and Genetic Characterization of Aeromonas hydrophila Phage AhMtk13a and Evaluation of Its Therapeutic Potential on Simulated Aeromonas Infection in Danio rerio.

Phage therapy can be an effective alternative to standard antimicrobial chemotherapy for control of Aeromonas hydrophila infections in aquaculture. Aeromonas hydrophila-specific phages AhMtk13a and AhMtk13b were studied for basic biological properties and genome characteristics. Phage AhMtk13a (Myovirus, 163,879 bp genome, 41.21% CG content) was selected based on broad lytic spectrum and physiologic parameters indicating its lytic nature. The therapeutic potential of phage AhMtk13a was evaluated in experimental studies in zebrafish challenged with A. hydrophila GW3-10 via intraperitoneal injection and passive immersion in aquaria water. In experimental series 1 with single introduction of AhMtk13a phage to aquaria water at phage-bacteria ratio 10:1, cumulative mortality 44% and 62% was registered in fish exposed to phage immediately and in 4 h after bacterial challenge, correspondingly, compared to 78% mortality in the group with no added phage. In experimental series 2 with triple application of AhMtk13a phage at ratio 100:1, the mortality comprised 15% in phage-treated group compared to the 55% in the control group. Aeromonas hydrophila GW3-10 was not detectable in aquaria water from day 9 but still present in fish at low concentration. AhMtk13a phage was maintained in fish and water throughout the experiment at the higher concentration in infected fish.

Skin bacteria of rainbow trout antagonistic to the fish pathogen Flavobacterium psychrophilum.

Rainbow trout fry syndrome (RTFS) and bacterial coldwater disease (BCWD) is a globally distributed freshwater fish disease caused by Flavobacterium psychrophilum. In spite of its importance, an effective vaccine is not still available. Manipulation of the microbiome of skin, which is a primary infection gate for pathogens, could be a novel countermeasure. For example, increasing the abundance of specific antagonistic bacteria against pathogens in fish skin might be effective to prevent fish disease. Here, we combined cultivation with 16S rRNA gene amplicon sequencing to obtain insight into the skin microbiome of the rainbow trout (Oncorhynchus mykiss) and searched for skin bacteria antagonistic to F. psychrophilum. By using multiple culture media, we obtained 174 isolates spanning 18 genera. Among them, Bosea sp. OX14 and Flavobacterium sp. GL7 respectively inhibited the growth of F. psychrophilum KU190628-78 and NCIMB 1947T, and produced antagonistic compounds of < 3 kDa in size. Sequences related to our isolates comprised 4.95% of skin microbial communities, and those related to strains OX14 and GL7 respectively comprised 1.60% and 0.17% of the skin microbiome. Comparisons with previously published microbiome data detected sequences related to strains OX14 and GL7 in skin of other rainbow trout and Atlantic salmon.

USE OF TOPICAL TREATMENTS AND EFFECTS OF WATER TEMPERATURE ON WOUND HEALING IN COMMON CARP (CYPRINUS CARPIO).

Skin lesions are frequently diagnosed in fish medicine. Although systemic fish treatments exist, little is known about the efficacy of topical drugs on fish skin lesions. This study aimed to investigate the efficacy of medical-grade honey and silver sulfadiazine on skin lesions using common carp (Cyprinus carpio) as a model. Additionally, the effect of temperature on the wound healing process was evaluated. Punch biopsies were generated on six fish per treatment group under anesthesia. Treatment groups received one of the following topical medications after wounding: Dr. Nordyke's Wound Honey, MicroLyte Ag Vet, or SilvaSorb Gel. Nontreated positive control groups were similarly wounded but did not receive topical treatment. Fish were housed at 10°C to 13°C or 18°C to 21°C for 29 days. Macroscopic evaluation and image collection of wounds were performed on days 0, 4, 8, 12, 21, and 29 after wounding to compare changes in wound areas and inflammation over time. On day 29, tissue samples were collected for histologic analysis. From day 12 after wounding onward, wounds in positive controls maintained at 18°C to 21°C were significantly smaller (days 12, 21, and 29: P < 0.0001) compared with positive controls kept at 10°C to 13°C. There was an overall improvement in macroscopic appearance in honey-treated groups compared with positive controls on day 12 after wounding at 18°C to 21°C (P = 0.001), whereas with the use of Microlyte and Silvasorb, wounds had increased inflammation grades (P < 0.0001 and P < 0.0001, respectively) with enlarged wound areas (P < 0.0001 and P < 0.001, respectively) in comparison with positive controls on day 12 after wounding at 18°C to 21°C. This study suggests that topical use of medical-grade honey produces positive effects on wound healing in the carp model and higher water temperatures enhance the effects, whereas the use of silver sulfadiazine and lower water temperatures delays or worsens the wound healing process.

Updating specific PCR primer for detection of Cryptocaryon irritans from reared Larimichthys polyactis.

Artificial breeding of small yellow croaker (Larimichthys polyactis) was recently achieved, providing a bright future for its commercial farming. In May 2019, a disease outbreak occurred among small yellow croakers in an aquaculture farm near Xiangshan Bay, charactering by white spots spotted on the surface of fish skin, gills and fins. The parasite was preliminarily identified as Cryptocaryon irritans based on morphological feature of the parasite and the symptoms on fish. However, the previously published specific primer pairs failed to confirm the existence of C. iriitans. Six nucleotides mismatches were discovered after mapping specific forward primer back to targeted gene. Therefore, an updated PCR specific primer was developed within the 9th highly variable region of 18S rRNA gene and conserved in all C. irritans sequences available in GenBank database. The specificity was verified in silico by Primer-BLAST against GenBank nucleotide. Laboratory cultured ciliates (Mesanophrys, Pseudokeronopsis and Uronema) as well as natural microbial community samples collected from sea water and river water was used as negative control to verify the specificity of the primer in situ. Besides, tank transfer method was used to evaluate the treatment of the parasite infection. By tank transfer method, 2.00 ± 0.61 out of 10 fish that already sever infected were successfully survived after 8 days treatment, meanwhile the control group died out at d 6. More loss to the treatment group during first five days was observed and may attribute to the combined effect from infection and stress the recent domesticated fish suffered during rotation. Therefore, tank transfer method was also effective to prevent small yellow croaker from further infection, however the loss of the small yellow croaker suffered from stress during rotation also needs to be carefully concerned. In conclusion, this study reported the first diagnose of C. irritans infection on small yellow croaker, provided updated specific primer to detect C. irritans infection on fish body and reported the effect of tank transfer on small yellow croaker treatment.

Geriatric Freshwater and Marine Fish.

As pain management finally becomes accepted for this last of the vertebrate taxa, fish medicine is finally reaching the sophistication of other vertebrates. The diseases of aging fish in captivity therefore need to be addressed. The degenerative organ/tissue changes and neoplasias of fish deserve the same diagnosis and treatments of their terrestrial counterparts including pain relief, anti-inflammatory medications, chemotherapy, surgery, joint supplements, regenerative cell therapy, and photobiomodulation. Besides the challenges of an aquatic environment, recognizing normal changes in older fish will be addressed in this article. Clinicians can appreciate the diversity of fishes and their unique anatomies, physiologies, and behaviors which translate to creative medicine.

Development of phage delivery by bioencapsulation of artemia nauplii with Edwardsiella tarda phage (ETP-1).

This study proposed that phage-enriched artemia could be a useful tool for transferring phage into the cultured fish (larvae or adult) as a feed, and introduce mode of phage administration and its safety in concern of tissue adaptation for efficient phage therapy in aquatic animals. First, whether Edwardsiella tarda phage (ETP-1) could attach or ingest by the artemia and optimum time period for the ETP-1 enrichment with artemia were investigated. ETP-1 dispersion, abundance and persistency, and zebrafish immune transcriptional responses and histopathology were evaluated after feeding the fish with ETP-1-enriched artemia. Hatched artemia nauplii (36 h) were enriched with 1.90 × 1011 PFUmL-1 of ETP-1, and maintained at 25 °C. The highest enrichment level was obtained after 4 h (3.00 × 109 PFUmL-1), and artemia were alive and active similar to control for 8 h. ETP-1 disseminated dose dependently to all the tissues rapidly (12 h). However, when feeding discontinued, it drastically decreased at day 3 with high abundance and persistency in the spleen (1.02 × 103) followed by the kidney (4.00 × 101) and the gut (1 × 101 PFUmL-1) for highest ETP-1-enriched artemia dose. In contrast, during continuous delivery of ETP-1-enriched artemia, ETP-1 detected in all the tissues (at day 10: gut; 1.90 × 107, kidney; 3.33 × 106, spleen; 5.52 × 105, liver; 6.20 × 104 PFUmL-1mg-1 tissues). Though the phage abundance varied, results indicated that oral fed ETP-1-enriched artemia disperse to the neighboring organs, even the absence of host as phage carrier. Non-significant differences of immune transcriptional and histopathology analysis between ETP-1-enriched artemia fed and controls suggest that no adverse apparent immune stimulation in host occurred, and use of ETP-1 at 1011 PFUmL-1 was safe. With further supportive studies, live artemia-mediated phage delivery method could be used as a promising tool during phage therapy against pathogenic bacteria to control aquatic diseases.

Transcriptome of Sphaerospora molnari (Cnidaria, Myxosporea) blood stages provides proteolytic arsenal as potential therapeutic targets against sphaerosporosis in common carp.

BACKGROUND: Parasites employ proteases to evade host immune systems, feed and replicate and are often the target of anti-parasite strategies to disrupt these interactions. Myxozoans are obligate cnidarian parasites, alternating between invertebrate and fish hosts. Their genes are highly divergent from other metazoans, and available genomic and transcriptomic datasets are limited. Some myxozoans are important aquaculture pathogens such as Sphaerospora molnari replicating in the blood of farmed carp before reaching the gills for sporogenesis and transmission. Proliferative stages cause a massive systemic lymphocyte response and the disruption of the gill epithelia by spore-forming stages leads to respiratory problems and mortalities. In the absence of a S. molnari genome, we utilized a de novo approach to assemble the first transcriptome of proliferative myxozoan stages to identify S. molnari proteases that are upregulated during the first stages of infection when the parasite multiplies massively, rather than in late spore-forming plasmodia. Furthermore, a subset of orthologs was used to characterize 3D structures and putative druggable targets. RESULTS: An assembled and host filtered transcriptome containing 9436 proteins, mapping to 29,560 contigs was mined for protease virulence factors and revealed that cysteine proteases were most common (38%), at a higher percentage than other myxozoans or cnidarians (25-30%). Two cathepsin Ls that were found upregulated in spore-forming stages with a presenilin like aspartic protease and a dipeptidyl peptidase. We also identified downregulated proteases in the spore-forming development when compared with proliferative stages including an astacin metallopeptidase and lipases (qPCR). In total, 235 transcripts were identified as putative proteases using a MEROPS database. In silico analysis of highly transcribed cathepsins revealed potential drug targets within this data set that should be prioritised for development. CONCLUSIONS: In silico surveys for proteins are essential in drug discovery and understanding host-parasite interactions in non-model systems. The present study of S. molnari's protease arsenal reveals previously unknown proteases potentially used for host exploitation and immune evasion. The pioneering dataset serves as a model for myxozoan virulence research, which is of particular importance as myxozoan diseases have recently been shown to emerge and expand geographically, due to climate change.

Use of bacteriophage vB_Pd_PDCC-1 as biological control agent of Photobacterium damselae subsp. damselae during hatching of longfin yellowtail (Seriola rivoliana) eggs.

AIMS: This study describes the effect of phage therapy on hatching of longfin yellowtail (Seriola rivoliana) eggs challenged with Photobacterium damselae subsp. damselae. METHODS AND RESULTS: A lytic phage (vB_Pd_PDCC-1) against P. damselae subsp. damselae was isolated and characterized. The use of phage vB_Pd_PDCC-1 increased the hatching rate of eggs, and reduced presumptive Vibrio species to non-detectable numbers, even in non-disinfected eggs. High-throughput 16S rRNA gene sequencing analysis revealed that phage vB_Pd_PDCC-1 caused significant changes in the composition and structure of the associated microbiota, allowing that members (e.g. those belonging to the family Vibrionaceae) of the class Gammaproteobacteria to be displaced by members of the class Alphaproteobacteria. CONCLUSIONS: To the best of our knowledge, this represents the first study evaluating phage therapy to control potential negative effects of P. damselae subsp. damselae during hatching of longfin yellowtail eggs. SIGNIFICANCE AND IMPACT OF THE STUDY: The Seriola genus includes several important commercial fish species due to its rapid growth and easy adaptability to confinement conditions. However, bacterial infections (especially those caused by Vibrio and Photobacterium species) are among the main limiting factors for the intensification of marine fish aquaculture, particularly during early development stages. Therefore, the use of phages, which are natural killers of bacteria, represents a promising strategy to reduce the mortality of farmed organisms caused by pathogenic bacteria.

Effects of Lactobacillus rhamnosus ATCC 7469 on Different Parameters Related to Health Status of Rainbow Trout (Oncorhynchus mykiss) and the Protection Against Yersinia ruckeri.

In the current study, we investigated the effect of a probiotic bacterium (Lactobacillus rhamnosus ATCC 7469) microencapsulated with alginate and hi-maize starch and coated with chitosan on improving growth factors, body composition, blood chemistry, and the immune response of rainbow trout (initial weight: 18.41 ± 0.32 g). Four experimental diets were formulated to feed fish for 60 days. They were control diet without any additive (C), diet added with beads without probiotic (E), a probiotic sprayed to the diet (L.r), and encapsulated probiotic supplemented diet (E-L.r). The results indicated that feeding with E-Lr significantly improved weight gain (84.98 g) and feed conversion ratio (0.95) compared to the other groups (P < 0.05). Also, fish fed E-Lr diet had a significantly higher value of whole-body protein (17.51%), total protein in the blood (4.98 g/dL), lysozyme (30.66 U/mL), alternative complement pathway hemolytic activity (134 U/mL), superoxide dismutase (203 U/mg protein), and catalase (528.33 U/mg protein) (P < 0.05) as compared to those fed the control diet. Similarly, a higher relative expression of immune-related genes such as interleukin-1 (Il-1) and tumor necrosis factor-alpha (TNF-1α) were reported in those fed E-L.r and L.r diets respectively. Interestingly, the fish fed dietary E-L.r had a significantly lower value of lipid in the whole body (4.82%) and cholesterol in the blood (160.67%) in comparison with those fed the control diet (P < 0.05). At the end of the experiment, all groups were challenged by Yersinia ruckeri where the survival rate of rainbow trout fed dietary E-L.r (70.36%) was statistically higher than that of the others (P < 0.05). Overall, the results suggested that encapsulated probiotic Lact. rhamnosus ATCC 7469 acted better than unencapsulated probiotic and has a potential to improve growth performance, flesh quality, and the immune response of rainbow trout.

Current status of aquatic veterinary services for ornamental fish in Australasia.

The ornamental fish industry is growing quickly globally. Asia represents 51% of the export market for this industry and in Singapore alone, it has an estimated market value of US$66 million. Despite the economic importance of this industry for Australasia, aquatic veterinary support for the ornamental fish sector is scarce. In many cases, ornamental fish owners do not seek advice from veterinarians. While the reasons behind this are difficult to elucidate, the low perceived value of fish compared to the cost of consulting a veterinarian, and limited knowledge about the availability of veterinary services for aquatic animals may contribute to the lack of interaction with veterinarians. Considering the growing importance of the ornamental fish industry, more education and data are needed about the services offered by veterinarians with aquatic animal health expertise. Knowledge on aquatic veterinary medicine is limited, reinforcing the need for more educational programmes and dissemination of aquatic veterinary services within the Australasian region.

Isolation and characterization of phage (ETP-1) specific to multidrug resistant pathogenic Edwardsiella tarda and its in vivo biocontrol efficacy in zebrafish (Danio rerio).

Edwardsiella tarda phage (ETP-1) was isolated from marine fish farm water to characterize its effect against pathogenic multidrug-resistant E. tarda. According to transmission electron microscopy results, ETP-1 is classified as a member of the family Podoviridae. ETP-1 showed MOI dependent E. tarda growth inhibition, a latent period of 60 min, and burst size of 100 PFU per infected cells. In host range tests, five out of eight E. tarda strains were sensitive to ETP-1 which had efficiency of plating index in the range 1-1.28. ETP-1 was stable over a broad range of pH and temperature. The size of the ETP-1 genome was predicted to be approximately 40 kb. Zebrafish exposed to ETP-1 showed no adverse gene responses to the inflammatory mediator cytokines, il1-ß, tnf-α, il-6, and il-10, the chemokine, cxcl-8a, and reactive oxygen species, sod-1. When zebrafish were bath exposed to ETP-1 for 12 days and simultaneously challenged with E. tarda (1.08 × 105 CFU fish-1), the survival rate was higher in phage exposed fish (68%) compared to that of the control (18%) until 4 days post challenge. Our results suggest that ETP-1 can be used as a potential bio-therapeutic candidate to control multi-drug resistant E. tarda infection in aquaculture.

Isolation and characterization of phage AHP-1 and its combined effect with chloramphenicol to control Aeromonas hydrophila.

To develop an alternative bio-control measure for multi-drug resistant pathogenic Aeromonas hydrophila, which causes motile Aeromonas septicemia in fish, novel virulent phage (AHP-1) was isolated from carp tissues. Morphological analysis by transmission electron microscopy revealed that AHP-1 belongs to Myoviridae family. AHP-1 displayed 81% of moderate adsorption by 25 min, and latent period of 40 min with burst size of 97 PFU mL-1 at an optimal multiplicity of infection (MOI) 0.1. AHP-1 was stable over a broad range of pH (4-11), temperature (4-50 °C), and salinity (0.1-3.5%). Both time and MOI dependent in vitro A. hydrophila growth inhibition was observed with AHP-1. AHP-1 (10 MOI) showed higher growth inhibition against A. hydrophila than chloramphenicol (5 µg mL-1), and combined treatment was more promising than individuals. Immune gene expression analysis of zebrafish upon continuous bath exposure to AHP-1 resulted significantly higher (il-6 and sod-1) or slight induction (tnf-α, il1-ß, il-10, and cxcl-8a) than controls at beginning of the phage exposure, but those lowered to basal level by day 12 post-phage exposure. It suggests no adverse immune responses have occurred for the AHP-1 dose that used, and have potential for the phage therapy. Further detailed in vivo studies are needed to confirm the protective efficacy of newly isolated AHP-1 against A. hydrophila infection.

Renal Disease in Teleost Patients.

The number of fish as pets far exceeds the populations of any other companion animal. As our knowledge of aquatic animal species and aquatic animal medicine continues to expand, veterinary expertise is becoming more critical to the client, researcher, fisheries biologist, aquarist, farmer, and fish hobbyist. Similar to other vertebrates, fish are susceptible to infectious and noninfectious renal disease. This article compares vertebrate renal anatomy and physiology and highlights some renal disease examples.

Bacteriophage Adherence to Mucus Mediates Preventive Protection against Pathogenic Bacteria.

Metazoans were proposed to host bacteriophages on their mucosal surfaces in a symbiotic relationship, where phages provide an external immunity against bacterial infections and the metazoans provide phages a medium for interacting with bacteria. However, scarce empirical evidence and model systems have left the phage-mucus interaction poorly understood. Here, we show that phages bind both to porcine mucus and to rainbow trout (Oncorhynchus mykiss) primary mucus, persist up to 7 days in the mucosa, and provide protection against Flavobacterium columnare Also, exposure to mucus changes the bacterial phenotype by increasing bacterial virulence and susceptibility to phage infections. This trade-off in bacterial virulence reveals ecological benefit of maintaining phages in the metazoan mucosal surfaces. Tests using other phage-bacterium pairs suggest that phage binding to mucus may be widespread in the biosphere, indicating its importance for disease, ecology, and evolution. This phenomenon may have significant potential to be exploited in preventive phage therapy.IMPORTANCE The mucosal surfaces of animals are habitat for microbes, including viruses. Bacteriophages-viruses that infect bacteria-were shown to be able to bind to mucus. This may result in a symbiotic relationship in which phages find bacterial hosts to infect, protecting the mucus-producing animal from bacterial infections in the process. Here, we studied phage binding on mucus and the effect of mucin on phage-bacterium interactions. The significance of our research is in showing that phage adhesion to mucus results in preventive protection against bacterial infections, which will serve as basis for the development of prophylactic phage therapy approaches. Besides, we also reveal that exposure to mucus upregulates bacterial virulence and that this is exploited by phages for infection, adding one additional layer to the metazoan-bacterium-phage biological interactions and ecology. This phenomenon might be widespread in the biosphere and thus crucial for understanding mucosal diseases, their outcome and treatment.

Grape pomace flour alleviates Pseudomonas aeruginosa-induced hepatic oxidative stress in grass carp by improving antioxidant defense.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen in aquaculture systems being associated to extensive liver damage caused by oxidative stress in both marine and freshwater fish. Dietary supplementation with natural antioxidants is considered a rational strategy to prevent hepatic diseases involved with oxidative stress. Bio-residues resulting from the wine industry, such as grape pomace, are potential sources of bioactive phenolic compounds that can be applied as supplement for animal production. Thus, the aim of this study was to evaluate whether dietary supplementation with grape pomace flour (GPF) was able to prevent or reduce the hepatic oxidative damage of grass carp, Ctenopharyngodon idella, experimentally infected by P. aeruginosa. Hepatic reactive oxygen species (ROS), metabolites of nitric oxide (NOx), thiobarbituric acid reactive substances, and protein carbonylation levels were higher in fish experimentally infected by P. aeruginosa compared to the control group. Hepatic superoxide dismutase and catalase activities and antioxidant capacity against peroxyl radical levels were also higher in fish experimentally infected by P. aeruginosa compared to the control group. Dietary supplementation with 300 mg/kg GPF prevented all alterations elicited by P. aeruginosa, with the exception of protein carbonylation levels. The dietary supplementation with 150 mg/kg GPF was not able to avoid alteration of the analyzed variables, being results similar to those infected (positive control). Based on these results, dietary supplementation with 300 mg/kg GPF prevented P. aeruginosa-induced liver damage in grass carp, and this protective effect occurred through prevention on excessive ROS and NOx production, as well as via prevention of lipid damage. Moreover, 300 mg/kg GPF exerted its hepatoprotective effects by improving enzymatic and non-enzymatic antioxidant defense system. In summary, this supplementation can be an interesting approach to prevent P. aeruginosa-induced liver damage.

Recent biocontrol measures for fish bacterial diseases, in particular to probiotics, bio-encapsulated vaccines, and phage therapy.

The objective of this review is to control fish bacterial diseases or infections through application of some promising novel biocontrol methods, such as probiotics, bio-encapsulated vaccines, and phage therapy, to avoid the disadvantages of traditional one that potentially affects fish and human health. Bacterial infection in intensive fish farming causes mass mortalities and the treatment of that requires the intensive use of chemicals and antibiotics. Several methods have been tried to control fish diseases including the use of antibiotics, but their haphazard use is associated with potentially negative effects as drug resistance and drug residues. The use of probiotics as biocontrol agents for aquaculture is increasing with the demand for environmental beneficial, eco-friendly alternatives for sustainable aquaculture production. The benefits of such supplements include improved food value, inhibition of pathogenic microorganisms, and increased immune response. The bio-encapsulated vaccine appears to be the most attractive method for releasing of vaccines. Several bioactive molecules which are specific for some diseases have been successfully encapsulated with nanoparticles in order to enhance their availability, bioactivity, and controlled delivery. Recently, "reverse vaccine" by using bio-informatics that aids in designing vaccines against infectious pathogens that are difficult to design, especially the intracellular bacteria. Additionally, the use of bacteriophages for biological control of pathogens in cultured fish has gained much interest. Several bacteriophages have been isolated specific to various pathogenic bacteria. Oral administration of phage cocktail is the most suitable way of application in fish, especially when large number of infected fish should be manipulated. Hence, in the following paragraphs, we will discuss some promising novel biocontrol methods that target the fish pathogens like probiotics, bio-encapsulated vaccines, and phage therapy.

RNase1 alleviates the Aeromonas hydrophila-induced oxidative stress in blunt snout bream.

RNase1 is an enzyme important in host defense in vertebrates where it degrades the RNA of bacteria and viruses. We evaluated the effect of RNase1 on the resistance to Aeromonas hydrophila infection in Megalobrama amblycephala. The fish were randomly divided into four groups: a blank group (none-treated M. amblycephala), a control group (injected PBS), a challenge group (A. hydrophila-injected) and a treatment group (pre-treated with RNase1 24 h before the A. hydrophila injection), and we collected five tissues of each group. Then we recorded changes in the levels of glutathione (GSH), oxidized glutathione (GSSG), hepatic catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) and lysozyme; and the relative mRNA expression of catalase (CAT), selenium-dependent glutathione peroxidase (GPx), Cu/Superoxide dismutase (Cu/Zn-SOD), glutamate-cysteine ligase (GCLC), glutathione reductase (GR) and nuclear factor erythroid 2-related factor 2 (Nrf2) for four groups. The expression of six genes was highest in liver and blood of the blank group. It was significantly higher in the gut of the treatment group (compared to control and challenge groups) 12 h after the infection. The treatment group exhibited a significant increase in GSH, SOD and CAT activity, and a decrease in GSSG, MDA and lysozyme content (compared to the control and challenge groups) 6 and 12 h after infection. These results suggest that supplementation with RNase1 protein can enhance resistance against A. hydrophila infections in M. amblycephala.