Response of the intestinal mucosal barrier of carp (Cyprinus carpio) to a bacterial challenge by Aeromonas hydrophila intubation after feeding with β‐1,3/1,6‐glucan

The effect of dietary β‐glucan on the bacterial community in the gut of common carp (Cyprinus carpio) was examined after oral application of Aeromonas hydrophila. Carp received either feed supplemented with 1% MacroGard^®, a β‐1,3/1,6‐glucan, or a β‐glucan‐free diet. Fourteen days after feeding, half of the carp from each group were intubated with 10⁹ colony‐forming units (CFU) of a pathogenic strain of A. hydrophila. Gut samples were taken 12 hr to 7 days after application and analysed using microbiological and molecular biological techniques (NGS, RT‐PCR‐DGGE). The reaction of the mucosa and the microbiota to an A. hydrophila intubation differed in carp fed with β‐glucan compared to carp from the control group. In β‐glucan fed carp, the total bacterial amount was lower but the number of bacterial species was higher. Bacterial composition was different for carp from both treatment groups. The number of mucin filled goblet cells was reduced in carp fed the β‐glucan diet. Mucus was obviously released from the goblet cells and was probably washed out of the gut together with high numbers of bacteria. This might be protective against pathogenic bacteria and, therefore, feeding with β‐glucan may provide protection against infections of the gut in carp.     

Flavobacteria colonizing the early life stages of hatchery‐incubated Chinook salmon Oncorhynchus tshawytscha (Walbaum 1792) are markedly diverse

Flavobacterial diseases are significant impediments to hatchery‐based fishery conservation and aquaculture productivity worldwide. Recent studies revealed a multitude of novel flavobacteria within the reproductive fluids and unfertilized eggs of feral Chinook salmon Oncorhynchus tshawytscha broodstock, some of which were associated with systemic disease. Herein, embryonated eggs/fry from these broodstock were assayed for flavobacteria while in incubator stacks in three hatcheries over 2 years, as was the water entering hatchery incubators. Overall, >65% of sampled eggs and 38% of fry were colonized by flavobacteria. One hundred and ninety‐one egg and fry‐associated flavobacterial isolates were characterized phenotypically and via 16S rRNA gene sequencing and phylogenetic analyses, revealing that the majority fell into 22 clades (i.e., 15 Flavobacterium spp. groups and seven Chryseobacterium spp. groups) that varied in presence by facility. Although some matched previously described fish‐pathogenic species, the majority were distinct from all described flavobacteria and likely represent novel species. Of concern, iodophor disinfection at the commonly utilized dose/duration for egg‐surface disinfection did not eliminate flavobacteria. Results also implicated maternal routes of infection and source water for some flavobacteria. In total, study findings underscore the complexity of flavobacterial ecology within hatchery environments and highlight the need for improved hatchery biosecurity practices.     

Enhanced growth and retarded gonadal development of farmed rainbow trout,Oncorhynchus mykiss (Walbaum) following a long‐day photoperiod

Long‐day photoperiods are considered as an effective managerial tool in manipulating of reproduction and somatic growth in a number of fish species. In this study, the effects of three different artificial photoperiods on the gonadal development and somatic growth of rainbow trout (Oncorhynchus mykiss L.) were investigated. Two years‐old immature female rainbow trout (279.94 ±2.25 g) were exposed to three artificial photoperiod regimes of 24L:0D, 18L:6D and 6L:18D and natural light (NL) regime for 5 months. The highest gonadosomatic indices were recorded in NL and 6L:18D groups while the rates were significantly lower in fish maintained under 18L:6D and 24L:0D photoperiods (P < 0.05). Mean oocyte diameters in fish exposed to 24L:0D and also to 18L:6D were significantly lower than the 6L:18D and NL groups. Photoperiods with 24L:0D and 18L:6D regimes resulted in significantly higher mean final weights and specific growth rates (SGR) than NL regime. The highest mean final weight (635.45 ± 16.19 g) and SGR (1.03 ± 0.04% day^?1) were obtained under 24L:0D photoperiod. Fish exposed to 24L:0D and 18L:6D showed the highest condition factor as 1.44 ± 0.01 and 1.44 ± 0.02 respectively, when compared with the NL (1.27 ± 0.01) and 6L:18D (1.34 ± 0.02) groups. Basically, the results suggested that continuous artificial lightning can be used as an influential factor in delaying gonadal development and enhancing somatic growth in rainbow trout during gonadal growth phase.     

Association of a specific major histocompatibility complex class IIβ single nucleotide polymorphism with resistance to lactococcosis in rainbow trout,Oncorhynchus mykiss (Walbaum)

Major histocompatibility complex (MHC) loci encode glycoproteins that bind to foreign peptides and initiate immune responses through their interaction with T cells. MHC class II molecules are heterodimers consisting of α and β chains encoded by extremely variable genes; variation in exon 2 is responsible for the majority of observed polymorphisms, mostly concentrated in the codons specifying the peptide‐binding region. Lactococcus garvieae is the causative agent of lactococcosis, a warm‐water bacterial infection pathogenic for cultured freshwater and marine fish. It causes considerable economic losses, limiting the profitability and development of fish industries in general and the intensive production of rainbow trout, Oncorhynchus mykiss (Walbaum), in particular. The disease is currently controlled with vaccines and antibiotics; however, vaccines have short‐term efficacy, and increasing concerns regarding antibiotic residues have called for alternative strategies. To explore the involvement of the MHC class II β‐1 domain as a candidate gene for resistance to lactococcosis, we exposed 400 rainbow trout to naturally contaminated water. One single nucleotide polymorphism (SNP) and one haplotype were associated with resistance (P < 0.01). These results are promising for using MHC class IIβ as a molecular marker in breeding rainbow trout resistant to lactococcosis.     

Dietary β‐glucan modulate haematological parameters,cytokines and gene expression in TLR and ERK pathways of rainbow trout (Oncorhynchus mykiss) during infection by Aeromonas salmonicida

To explore the role of β‐glucan (0, 0.05%, 0.1% and 0.2%) in resisting bacteria, rainbow trout (Oncorhynchus mykiss) was challenged with Aeromonas salmonicida after 42‐day β‐glucan administration, and then sampled at 0, 2, 4 and 6 days post infection (dpi). The data showed that 0.2% β‐glucan reduced the accumulated mortality rates compared with the ICG (infected control group) (p < .05). The white blood cells, red blood cells and haemoglobin were higher in the 0.2% β‐glucan group (BG) than the ICG (p < .05). 0.1% and 0.2% β‐glucan elevated serum total antioxidative capability and glutathione activity but alleviated the increase of serum alkaline phosphatase activity and glucose concentration (p < .05) during infection. Serum TNF‐α, IL‐1β, IL‐8 and IgM in three BGs elevated remarkably on 6 dpi compared with the ICG (p < .05). Expression of tnf, il1b and cxcl8 of the head kidney in the 0.1% and 0.2% BGs was higher than the ICG on 4 dpi while ighm expression in the 0.2% BG was higher than in the ICG on 2 and 6 dpi (p < .05). 0.1% and 0.2% β‐glucan increased the expression of tlr5m, tlr5s, tmek and myd88 in the spleen after infection (p < .05). Similarly, 0.2% β‐glucan up‐regulated the expression of tmek, myd88, oncmyk‐dab and c3 in head kidney (p < .05). Overall, 0.2% dietary β‐glucan effectively decreased accumulated mortality rate by modulating the biochemistry process, cytokines, and activating TLR and ERK signalling pathways during A. salmonicida infection.     

In vitro and in vivo antifungal activity of Satureja cuneifolia ten essential oil on Saprolegnia parasitica strains isolated from rainbow trout (Oncorhynchus mykiss,Walbaum) eggs

In the present study, the chemical composition and the antifungal properties against Saprolegnia parasitica (in vitro and in vivo) of the essential oils of thyme (Satureja cuneifolia) from Mediterranean region of Turkey were evaluated for the first time. The composition of oils was analysed using gas chromatography/mass spectrometry (GC/MS). The major constituents of oil of S. cuneifolia were cavracrol (46,84%) and cymene (16.90%). Antifungal effects of S. cuneifolia essential oil against S. parasitica strains (A1 and E1) were detected by disc diffusion and tube dilution assays. The antifungal effect of S. cuneifolia was determined to be stronger against S. parasitica E1 isolate (MIC 50 μL mL^?1, MLC 250 μL mL^?1) compared with S. parasitica A1 isolate (MIC 50 μL mL^?1, MLC 500 μL mL^?1). Following in vitro assays, effective doses of S. cuneifolia for disease control in rainbow trout eggs experimentally infected with S. parasitica were investigated. For this aim, infected eggs were treated with the essential oil (0, 5, 10, 20 and 50 ppm) during incubation period (21 days) after fertilization. Formalin (5 mL L^?1) was used as positive control. Hatching rate of eggs at the end of incubation period were calculated. The highest hatching rates were recorded in S. parasitica E1 strain at 5 and 10 ppm concentrations of S. cuneifolia and in S. parasitica A1 strain at 10 and 20 ppm (P < 0.05).     

1,3‐1‐6 ß‐glucans enhance tissue regeneration in zebrafish (Danio rerio): Potential advantages for aquaculture applications

High aquacultural rearing density and handling of fish may frequently result in skin or gills wounds, thereby facilitating the onset of secondary infections. The capacity of the zebrafish to regenerate tissues, as well as fins and other organs, makes it an ideal animal model for studying the mechanisms of tissue regeneration. Since macrophages are involved in tissue regeneration, a diet including ß‐glucans might positively affect the process through activation of macrophages and other immune pathways. Consequently, the aim of this study was to investigate the effects of the oral administration of 1,3‐1,6 β‐glucans on the regeneration process of the caudal fin after its amputation in zebrafish. One hundred and twenty zebrafish were randomly distributed into four groups with three replicates each: an untreated non‐amputated group (CNA) and an untreated amputated group (CA) fed a control diet; two treated and amputated groups (MI and MII) fed for 14 days the same diet with the addition of two differently extracted 1,3‐1,6 ß‐glucans (MacroGard^® and Experimental MacroGard^®, Biorigin^©). ß‐glucans were added to allowed the administration of 12.5 mg/kg of fish body weight (0.35 g/kg of feed). Results showed that 1,3‐1,6 ß‐glucans decreased fish mortality rate and enhanced both daily and cumulative regenerated fin area, independent of the specific ß‐glucan extraction method used. Based on the mechanisms similarities of the innate immune system and tissue regeneration among different teleost species, these results may likely be extended to species of interest for the aquaculture sector.     

Pro‐inflammatory caspase‐1 activation during the immune response in cells from rainbow trout Oncorhynchus mykiss (Walbaum 1792) challenged with pathogen‐associated molecular patterns

In response to pathogens, the higher vertebrate innate immune system activates pro‐inflammatory caspase‐1 which is responsible for the processing and secretion of several important cytokines involved in the host's defence against infection. To date, caspase‐1 has been described in few teleost fish, and its activity has been demonstrated through substrate cleavage and inhibition by pharmacological agents. In this study, the detection of the active form of caspase‐1 during the immune response in salmonid fish is described, where two antibodies were produced. These antibodies differentially recognize the structural epitopes of the inactive pro‐caspase‐1 and the processed active form of the caspase. Firstly, caspase‐1 activation was demonstrated in vitro by ELISA, Western blotting and immunocytochemistry in rainbow trout macrophages exposed to different pathogen‐associated molecular patterns plus the pathogen Aeromonas hydrophila. This activity was clearly abrogated by a caspase inhibitor and seems to be unrelated to IL‐1β secretion. Caspase‐1 activation was then validated in vivo in gill cells from fish challenged with Aeromonas salmonicida. These results represent the first demonstration of caspase‐1 activation in salmonids, and the first evidence of the putative regulatory role which this protease plays in inflammatory response in this fish group, as described for some other teleosts and mammals.     

The effect of β‐1,3‐glucan derived from Euglena gracilis (Algamune™) on the innate immunological responses of Nile tilapia (Oreochromis niloticus L.)

Algamune^? is a commercial additive produced from Euglena gracilis, providing a rich source of the β‐1,3‐glucan paramylon. Isolated kidney phagocytes of Nile tilapia were incubated with graded doses (0, 8, 16, 32, 64 and 128 μg/ml) of Algamune^? and purified paramylon to gauge their ability to elicit the production of reactive oxygen species. A linear response was observed for extracellular superoxide anion for both sources but only Algamune^? for intracellular superoxide anion. After corroborating the immunostimulant properties ex vivo, a feeding trial was conducted to evaluate the dietary supplementation of Algamune^? (0, 100, 200, 400 and 800 mg/kg of diet) for Nile tilapia. Fish were fed for 3 weeks, after which, fish were sampled for blood and head kidney phagocytes. The remaining fish were challenged with Streptococcus iniae. Macrophage extracellular superoxide anion production was significantly elevated in fish fed diets with 200 mg of Algamune^? kg^?1 when compared to fish fed the basal diet. Even though the disease challenge did not show statistical differences, it is worth mentioning that fish fed intermediate doses of Algamune^? had lowest numerical mortality values. Therefore, Algamune^? was demonstrated to enhance some immunological responses of tilapia both in ex vivo and in vivo.     

Application of dietary supplements (synbiotics and probiotics in combination with plant products and β‐glucans) in aquaculture

This review addresses the dietary supplements, synbiotics and probiotics in combination with plant products or yeast/β‐glucans in aquaculture. The potential applications of synbiotics have a relatively short history in aquaculture, but have generated interest because of numerous benefits reported in endothermic animals. Since the first study was published by (Aquaculture Science in 2009) the concept has been used in aquaculture to reveal effects on growth performance, gut microbiota, gut histology, immune parameters, haematological and biochemical parameters as well as increased disease resistance. However, a limited number of probiotic bacteria (mainly Bacillus and Enterococcus) and prebiotics (mannan oligosaccharides, fructooligosaccharides, short‐chain fructooligosaccharides, galactooligosaccharides, arabinoxylan–oligosaccharides, isomaltooligosaccharide, chitosan oligosaccharide and inulin) have been used. Additionally, some studies have used plant products or yeast/β‐glucans in combination with probiotics, and these studies suggested that these dietary supplements promote growth performance and boost some immune parameters. The present review identifies evaluations of gut microbiota and gut morphology, and mucosal immune response as significant gaps in existing knowledge and suggests issues that merit further investigations to conclude the potential of dietary supplements in aquaculture.     

Phenotype of Aeromonas salmonicida sp. salmonicida cyclic adenosine 3′,5′‐monophosphate receptor protein (Crp) mutants and its virulence in rainbow trout (Oncorhynchus mykiss)

Precise deletion of genes related to virulence can be used as a strategy to produce attenuated bacterial vaccines. Here, we study the deletion of the cyclic‐3′,5′‐adenosine monophosphate (cAMP) receptor protein (Crp) in Aeromonas salmonicida, the aetiologic agent of furunculosis in marine and freshwater fish. The Crp protein is a conserved global regulator, controlling physiology processes, like sugar utilization. Deletion of the crp gene has been utilized in live attenuated vaccines for mammals, birds and warm water fish. Here, we characterized the crp gene and reported the effect of a crp deletion in A. salmonicida virulent and non‐virulent isolates. We found that A. salmonicida Δcrp was not able to utilize maltose and other sugars, and its generation time was similar to the wild type. A. salmonicida ?crp showed a higher ability of cell invasion compared to the wild type. Fish challenges showed that A. salmonicida ?crp is ~6 times attenuated in Oncorhynchus mykiss and conferred protective immunity against the intraperitoneal challenge with A. salmonicida wild type. We concluded that deletion of A. salmonicida crp influences sugar utilization, cell invasion and virulence. Deletion of crp in A. salmonicida could be considered as part of an effective strategy to develop immersion live attenuated vaccines against furunculosis.     

Characterization of β‐catenin 1 during the gonad development in the common carp (Cyprinus carpio)

β‐catenin gene is a pivotal gene for gonad development and maintenance of ovarian function in mammals. However, little is known about its expression and function in gonad development of fish. In this study, a complete cDNA (3342 bp) sequence of β‐catenin 1 was cloned from the common carp, Cyprinus carpio, by RACE PCR, which encodes a 780‐amino‐acid protein. Quantitative real‐time PCR demonstrated that β‐catenin 1 mRNA expressions were high in the testis and ovary tissue and the expression increased as the testes developed and the early stage ovaries developed. Western blot results revealed a single immunoreactive band with an estimated molecular weight of 90 kDa in testes. Immunohistochemistry analysis revealed that the β‐catenin 1 protein was concentrated mainly in the cytoplasm of early development stage of oocyte cells and in the cytomembrane of developing and mature sperm cells. 17β‐Ethinylestradiol injecting intraperitoneally into the fish decreased the relative β‐catenin 1 mRNA expression level except 1 μg/g 72 hr and 5 μg/g 48 hr of treatments in the ovary by real‐time PCR. These results suggest, for the first time, that β‐catenin 1 is an essential protein in gonad development and might be involved in ovarian early development of C. carpio.     

Biochemical parameters in the blood of gilthead sea bream (Sparus aurata Linnaeus, 1758) supplemented with commercially available β‐glucan‐based product (IMUNO‐2865®)

The aim of this study was to evaluate the effects of a promising new immune stimulant in aquaculture (IMUNO‐2865^®) on biochemical parameters in sea bream during the winter stress period. A total of 640 sea bream were fed throughout 90 days with diets containing 0 (Group 1), 1 (Group 2), 10 (Group 3) and 25 g (Group 4) of IMUNO‐2865^® kg^?1 of feed. Samples were taken each month and 90 days after supplementation. No statistical differences among treatment groups were noticed for the following biochemical parameters: glucose (GLU), aspartate aminotransferase (AST), plasma cholesterol (CHOL), lactate dehydrogenase (LDH), urea (URE) and creatinine (CREA). At the final sampling, total ammonia (NH₃) was higher in Groups 3 and 4 compared to the control and the low supplementation group (p < .05), while total proteins (TP) was significantly higher in Group 4 compared to all other groups, and in Groups 2 and 3 compared to the control group (p < .05). Blood Ca⁺⁺ levels were significantly higher after 60 days of feeding in all treatment groups compared to the control, and remained elevated in Group 4 even after 90 days following cessation of supplementation (p < .05). The results of this study described the increase of biochemical parameters in the blood of sea bream after use of IMUNO‐2865^® but future research is needed to evaluate its potentially immunostimulative effect on fish in commercial aquaculture.     

The effect of dietary protein source (fishmeal vs. plant protein) and non‐starch polysaccharide level on fat digestibility and faecal bile acid loss in rainbow trout (Oncorhynchus mykiss)

This study investigated in rainbow trout (Oncorhynchus mykiss) if diet composition and feeding level affect faecal bile acid loss, and whether this reflects on the apparent digestibility coefficient (ADC) of fat. Six diets were formulated with either fishmeal or plant protein as main protein source. This created a contrast in the supply of bile acids, the bile acid precursor cholesterol, taurine and the taurine precursors (methionine + cysteine) involved in bile acid conjugation. For both protein sources, three diets were formulated with increasing inclusion of a non‐starch polysaccharide (NSP)‐rich ingredient mixture (0.0, 82.0 and 164.2 g/kg diet). This aimed at enhancing faecal bile acid loss. Fish were fed both restrictively (1.2% BW/day) and to satiation. A similar fat ADC was found when substituting fishmeal with a plant protein mixture, suggesting that the lower content of bile acids, cholesterol, taurine, methionine and cysteine in the plant‐based diets did not limit fat digestion. Faecal bile acid loss increased alongside dietary NSP level, however, only during satiation feeding and most strongly for fish fed the fishmeal‐based diets. Enhanced faecal bile acid loss was not caused by NSP‐bile acid binding/entrapment, but by an increase in faeces production. During satiation feeding, fat ADC negatively correlated with faecal bile acid loss. From this it is concluded that bile acid availability/synthesis can become limiting for fat digestion in rainbow trout under conditions that enhance faecal bile acid loss (i.e. dietary NSP level and feeding level).     

Rainbow trout Oncorhynchus mykiss (Walbaum) type IV ice‐structuring protein LS‐12 in the acute‐phase response to Flavobacterium psychrophilum infection

A previous proteomic study examining the plasma acute‐phase response of rainbow trout to sterile inflammation highlighted an unidentified 9.5‐kDa spot using 2D‐PAGE, which was dramatically increased. The 15 amino acid sequence obtained from this protein spot allowed rapid amplification of cDNA ends PCR to generate a 443‐bp nucleotide sequence that was 98.6% similar to type‐4 ice‐structuring protein LS‐12 from Atlantic salmon Salmo salar Linnaeus. Quantitative reverse translation PCR and an ELISA were used to measure gene expression and plasma concentrations of LS‐12 following experimental intraperitoneal injection of rainbow trout with either 10⁶ or 10⁸ colony‐forming units (CFU) of Flavobacterium psychrophilum. There was no significant change in the plasma concentration of LS‐12 up to 15 days post‐infection in any group. Hepatic LS‐12 gene expression was significantly reduced at 3 and 6 days (p < 0.001) post‐infection in fish injected with 10⁸ CFU of F. psychrophilum relative to control fish, while branchial or head kidney expression was unchanged. Infected fish had significantly increased hepatic gene expression of serum amyloid A, confirming an acute‐phase response. Under the conditions used, LS‐12 is not a positive acute‐phase protein in rainbow trout.     

Effect of aromatable androgen (17‐methyltestosterone) on induced maturation of silver European eels (Anguilla Anguilla): Oocyte performance and synchronization

The reproductive performances of silver European eel in term of gonad development and egg production, employing slow‐release implants with the androgen 17‐MT (1 mg) in combination with traditional weekly injection of carp pituitary extract (CPE) was evaluated. Wild female European eels (Anguilla anguilla) underwent a standard induction protocol with CPE and were randomly divided into three groups (N‐group, no implant; Y‐group, with implant; and control, C‐group, no treatment). The results showed that 17‐MT‐treated females (Y‐group) reproduced spontaneously about 6 weeks earlier than the N‐group females with a saving of almost 40% in CPE and time of induction. Concerning artificial induction of maturation in female silver eels, our study demonstrated that they positively respond to androgen exposure also in terms of eggs productivity. Indeed, Y‐group was more productive than N‐group: in Y‐group, 11 eels ensured an eggs production that exceeded 50% of initial body weight (BW), whereas in N‐group only three eels have exceeded this value. The results suggest that 17‐MT should be considered in future protocols for the improvement of the artificial reproduction of female silver European eels.     

Epitheliocystis in lake trout Salvelinus namaycush (Walbaum) is associated with a β‐proteobacteria

Lake trout Salvelinus namaycush (Walbaum) raised for stocking experienced yearly (2011–13) winter epizootics of epitheliocystis. Affected fish were dispersed on the bottom of the tank, had decreased feed and fright response, and mortality often reached 40%. Peak mortality occurred within 3 weeks of the appearance of clinical signs, and outbreaks typically lasted 6 weeks. Affected fish had no gross lesions but histologically had branchial epithelial necrosis and lamellar hyperplasia, with small to large numbers of scattered epithelial cells containing 10‐ to 20‐μm inclusions. A longitudinal study was undertaken of one annual outbreak, and lamellar hyperplasia was most closely associated with mortality. The number of inclusions was statistically greater (P < 0.05) before and during peak mortality, but inclusions were present in low numbers before clinical signs occurred. Results of histochemical staining, immunohistochemistry and transmission electron microscopy supported the presence of a β‐proteobacteria rather than a Chlamydiales bacterium within inclusions. PCR primers to identify Chlamydiales did not give consistent results. However, the use of universal 16S rDNA bacterial primers in conjunction with laser capture microdissection of inclusions demonstrated that a β‐proteobacteria was consistently associated with affected gills and is more likely the cause of the disease in lake trout.