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.

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.

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.

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.

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.

Shark IgNAR-derived binding domains as potential diagnostic and therapeutic agents.

Many of the most successful drugs generated in recent years are based upon monoclonal antibodies (mAbs). However, for some therapeutic and diagnostic applications mAbs are far from ideal; for example, while their relatively large size and inherent receptor binding aids their longevity in vivo it can also limit their tissue penetration. Further, their structural complexity makes them expensive to produce and prone to denaturation in non-physiological environments. Thus, researchers have been searching for alternative antigen-binding molecules that can be utilized in situations where mAbs are suboptimal tools. One potential source currently being explored are the shark-derived binding domains known as VNARs. Despite their small size VNARs can bind antigens with high specificity and high affinity. Combined with their propensity to bind epitopes that are inaccessible to conventional mAbs, and their ability to resist denaturation, VNARs are an emerging prospect for use in therapeutic, diagnostic, and biotechnological applications.

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.

Dietary Withania sominefera root confers protective and immunotherapeutic effects against Aeromonas hydrophila infection in Nile tilapia (Oreochromis niloticus).

In the present study, effect of dietary Withania sominefera (W. sominefera) root powder was evaluated to modulate immune and antioxidant response against Aeromonas hydrophila (A. hydrophila) infection in Nile tilapia (Oreochromis niloticus). W. sominefera root powder supplemented diets at two concentrations 2.5% (W 2.5%) and 5% (W 5%); fed for 6 weeks prior to the A. hydrophila challenge and continued the same respective diets during the post challenge period (2 weeks). Results showed that fish fed W. sominefera at 5% enhanced immune response in both pre and post-challenge period. NBT level exhibited only significant increase (P < 0.05) in the pre-challenge period compared to control. Malondialdehyde (MDA) levels in liver and muscle revealed significant decrease in both Withania supplemented groups compared to the control in post challenge period. Antioxidant enzymes activities (catalase/CAT/, glutathione S-transferase/GST/, glutathione/GSH; and superoxide dismutase/SOD) were improved in liver and muscle in post challenge period. Glutathione peroxidase (GPx) level in muscle and serum total antioxidant capacity (TAC) showed a significant increase in both Withania supplemented groups compared to the control post challenge. Withania supplementation enhanced disease resistance against A. hydrophila and reduced mortalities (20%), especially at supplemented concentration of 5%. Our findings suggest that W. sominefera root powder may have protective and immunotherapeutic roles in Nile tilapia against A. hydrophila infection which may be useful in controlling important fish bacterial diseases.

Mycobacterium marinum infection in fish and man: epidemiology, pathophysiology and management; a review.

Mycobacterium marinum is an opportunistic pathogen inducing infection in fresh and marine water fish. This pathogen causes necrotizing granuloma like tuberculosis, morbidity and mortality in fish. The cell wall-associated lipid phthiocerol dimycocerosates, phenolic glycolipids and ESAT-6 secretion system 1 (ESX-1) are the conserved virulence determinant of the organism. Human infections with Mycobacterium marinum hypothetically are classified into four clinical categories (type I-type IV) and have been associated with the exposure of damaged skin to polluted water from fish pools or contacting objects contaminated with infected fish. Fish mycobacteriosis is clinically manifested and characterized in man by purple painless nodules, liable to develop into superficial crusting ulceration with scar formation. Early laboratory diagnosis of M. marinum including histopathology, culture and PCR is essential and critical as the clinical response to antibiotics requires months to be attained. The pathogenicity and virulence determinants of M. marinum need to be thoroughly and comprehensively investigated and understood. In spite of accumulating information on this pathogen, the different relevant data should be compared, connected and globally compiled. This article is reviewing the epidemiology, virulence factors, diagnosis and disease management in fish while casting light on the potential associated public health hazards.

VP2 (PTA motif) encoding DNA vaccine confers protection against lethal challenge with infectious pancreatic necrosis virus (IPNV) in trout.

IPNV in Atlantic salmon is represented by various strains with different virulence and immunogenicity linked to various motifs of the VP2 capsid. IPNV variant with P217, T221, A247 (PTA) motif is found to be avirulent in Atlantic salmon, but virulent in rainbow trout, and other salmonid species. This study describes a DNA vaccine delivered intramuscularly encoding the VP2 protein of infectious pancreatic necrosis virus (IPNV) with PTA motif that confers high protection in rainbow trout (Oncorhynchus mykiss). Intramuscular injection of 2, 5 and 10 µg of DNA (pcDNA3.1-VP2) in rainbow trout fry (4-5 g), confers relative protection of 75-83% in the different vaccine groups at 30 days post vaccination (450° days). The VP2 gene is expressed in spleen, kidney, muscle and liver at day 30 post-vaccination (RT-PCR), and IFN-1 and Mx-1 mRNA are upregulated at early time post vaccination, and so also for IgM, IgT, CD4 and CD8 in the head kidney of vaccinated fish compared to controls, 15 and 30 days post vaccination. Significant increase of serum anti-IPNV antibodies was found 30-90 days post-vaccination that was correlated with protection levels. Mortality corresponded with viral VP4 gene expression were significantly decreased in vaccinated and challenged fish. This shows for the first time that a VP2-encoding DNA vaccine delivered intramuscularly elicits a high level of protection alongside with high levels of circulating antibodies in rainbow trout and a lowered viral replication.

The fish parasite Ichthyophthirius multifiliis - Host immunology, vaccines and novel treatments.

Ichthyophthirius multifiliis, the causative agent of white spot disease (ichthyophthiriasis) is a major burden for fish farmers and aquarists globally. The parasite infects the skin and the gills of freshwater fish, which may acquire a protective adaptive immune response against this disease, making vaccine strategies feasible. However, there is no prophylactic treatment available and repetitive treatments with auxiliary substances are needed to control the infection. Historically, a variety of drugs and chemicals have been used to combat the disease but due to changing regulations and recognition of carcinogenic and environmentally damaging effects the most efficient compounds are prohibited. A continuous search for novel substances, which are highly effective against the parasites and harmless for the fish is ongoing. These compounds should be environmentally friendly and cost-effective. This review presents recent progress within host immunology, vaccinology and a description of novel substances, which have been tested as treatments against ichthyophthiriasis.

Bactericidal activity of fish galectin 4 derived membrane-binding peptide tagged with oligotryptophan.

Galectins belong to the family of galactoside-binding proteins which act as pathogen recognition receptors by recognizing and binding to the carbohydrate present in the bacterial membranes. In this study, a Galectin-4 sequence was identified from the constructed cDNA library of Channa striatus and its structural features were reported. Gene expression analysis revealed that CsGal4 was highly expressed in liver and strongly induced by Epizootic Ulcerative Syndrome (EUS) causing pathogens such as Aphanomyces invadans, Aeromonas hydrophila and a viral analogue, poly I:C. To understand the antimicrobial role of putative dimerization site of CsGal4, the region was chemically synthesized and its bactericidal effect was determined. G4 peptide exhibited a weak bactericidal activity against Vibrio harveyi, an important aquaculture pathogen. We have also determined the bactericidal activity of the dimerization site by tagging pentamer oligotryptophan (W5) at the C-terminal of G4 peptide. Flow cytometry analysis revealed that G4W induced drastic reduction in cell counts than G4. Electron microscopic images showed membrane blebbings in V. harveyi which indicated the membrane disrupting activity of G4W. Interestingly, both the peptides did not exhibit any hemolytic activity and cytotoxicity towards peripheral blood cells of Channa striatus and the activity was specific only towards the bacterial membrane. Our results suggested that addition of W5 at the C-terminal of membrane-binding peptide remarkably improved its membrane disrupting activity.

Fish Oncology: Diseases, Diagnostics, and Therapeutics.

The scientific literature contains a wealth of information concerning spontaneous fish neoplasms, although ornamental fish oncology is still in its infancy. The occurrence of fish neoplasms has often been associated with oncogenic viruses and environmental insults, making them useful markers for environmental contaminants. The use of fish, including zebrafish, as models of human carcinogenesis has been developed and knowledge gained from these models may also be applied to ornamental fish, although more studies are required. This review summarizes information available about fish oncology pertaining to veterinary clinicians.

Fighting fish parasites with photodynamically active chlorophyllin.

Water-soluble chlorophyll (chlorophyllin) was used in a phototoxic reaction against a number of fish ectoparasites such as Ichtyobodo, Dactylogyrus, Trichodina, and Argulus. Chlorophyllin is applied to the water at concentrations of several micrograms per milliliter for a predefined incubation time, and afterwards, the parasites are exposed to simulated solar radiation. Application in the dark caused only little damage to the parasites; likewise, light exposure without the addition of the photosensitizer was ineffective. In Ichthyobodo, 2 µg/mL proved sufficient with subsequent simulated solar radiation to almost quantitatively kill the parasites, while in Dactylogyrus, a concentration of about 6 µg/mL was necessary. The LD50 value for this parasite was 1.02 µg/mL. Trichodina could be almost completely eliminated at 2 µg/mL. Only in the parasitic crustacean Argulus, no killing could be achieved by a photodynamic reaction using chlorophyllin. Chlorophyllin is non-toxic, biodegradable, and can be produced at low cost. Therefore, we propose that chlorophyllin (or other photodynamic substances) are a possible effective countermeasure against several ectoparasites in ponds and aquaculture since chemical remedies are either forbidden and/or ineffective.

Viruses of fish: an overview of significant pathogens.

The growing global demand for seafood together with the limited capacity of the wild-capture sector to meet this demand has seen the aquaculture industry continue to grow around the world. A vast array of aquatic animal species is farmed in high density in freshwater, brackish and marine systems where they are exposed to new environments and potentially new diseases. On-farm stresses may compromise their ability to combat infection, and farming practices facilitate rapid transmission of disease. Viral pathogens, whether they have been established for decades or whether they are newly emerging as disease threats, are particularly challenging since there are few, if any, efficacious treatments, and the development of effective viral vaccines for delivery in aquatic systems remains elusive. Here, we review a few of the more significant viral pathogens of finfish, including aquabirnaviruses and infectious hematopoietic necrosis virus which have been known since the first half of the 20th century, and more recent viral pathogens, for example betanodaviruses, that have emerged as aquaculture has undergone a dramatic expansion in the past few decades.

Diagnostic techniques and treatments for internal disorders of Koi (Cyprinus carpio).

The most common problems that occur in koi involve external pathogens and environmental conditions. Techniques for external fish examination and water quality analysis have been well described in the veterinary literature. However, there are also some internal disorders of koi, such as gas bladder abnormalities affecting the fish's buoyancy, neoplasia, egg binding (roe retention), and spinal disorders that can be diagnosed with common veterinary medical procedures. Diagnostic techniques along with available treatments for these disorders are presented in this article.

Effect of Lactococcus lactis CLFP 100 and Leuconostoc mesenteroides CLFP 196 on Aeromonas salmonicida Infection in brown trout (Salmo trutta).

Aeromonas salmonicida is the etiological agent of furunculosis in salmonid fish. This pathogen is important from an epizootic perspective because fish surviving an outbreak can remain lifelong asymptomatic carriers, serving as reservoirs of infection. As a result, the early detection and the control of infection are essential to prevent the spread of new furunculosis outbreaks. We have thus analyzed the effect of probiotic administration on the incidence of A. salmonicida in brown trout (Salmo trutta), that were subjected to temperature stress. Treatment with probiotic strains (Lactococcus lactis CLFP 100 and Leuconostoc mesenteroides CLFP 196) resulted in a higher survival rate after challenge, activation of phagocytic cells in the head kidney, and a lower rate of pathogen proliferation in the intestine as determined by real-time PCR.

Neoplasia in fishes.

Similar to higher vertebrates, neoplasia is not an uncommon disease in fishes, which are the largest group of vertebrates. However,neoplasia in fishes is generally a benign condition with relatively few exceptions of malignant disease. The objective of this discussion is to provide an overview of neoplasia and the various neoplastic disease conditions in fishes according to organ system,including the few neoplasms of species that are familiar to the aquatic animal or exotic animal practitioner. The discussion also considers the various nonneoplastic lesions in fishes that may be confused with neoplasms, and treatment of neoplastic disease in fishes that is generally restricted to surgical intervention.

The effectiveness of adenoviral vectors to deliver and express genes in rainbow trout, Oncorhynchus mykiss (Walbaum).

The efficacy of adenoviral vectors for gene delivery into fish cells, both in vitro and in vivo, was evaluated. Vectors utilized were of human adenovirus serotype 5 (Ad), which are commonly used in human clinical trials, but have not been assessed for gene delivery to fish. Because nothing is known about Ad receptors in fish, both an Ad (Ad5Luc1) with natural tropism for the coxsackie and adenovirus receptor (CAR), as well as an infectivity enhanced Ad (Ad5LucRGD) were included within this study. Gene expression was detected in cell lines using either vector. The levels seen with Ad5LucRGD were much higher than for Ad5Luc1 in most lines except CHSE-214. Transduction of CHSE-214 cells with Ad5Luc1 could be blocked with an excess of a competitive inhibitor, suggesting that these cells possess a CAR homologue thatmediates attachment of Ad, similar to that seen in mammalian cells. In vivo gene delivery was attempted by several methods, with significant expression seen only via intramuscular injection, although infection efficiency was low. Thus it was observed that several teleost cell lines are capable of being infected and one cell line expressed a human serotype adenoviral receptor homologue that aids in Ad infection. Additionally, in vivo studies indicated that muscle tissue of rainbow trout could be infected with Ad vectors, suggesting an alternative gene delivery strategy for this animal.