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Carassius auratus gibelio, commonly known as crucian carp, is the main species of freshwater aquaculture in China. Due to high-density aquaculture and environmental pollution, various diseases continue to emerge, especially the diseases caused by pathogenic bacteria have seriously restricted the development of freshwater aquaculture. Among these, Aeromonas hydrophila (AH) is the one of the most serious fish disease causing economic losses among fish farmers. An in-depth understanding of the crucian carp's immune system and its genes involved in the response to bacterial infection is necessary for disease control. In this study, the breeding period was divided into the control group (C group) and the Bacillus licheniformis (B. licheniformis) group (BL group). After 4 weeks of culture, the crucian carp was infected with AH and divided into the control group (Cg group) and the B. licheniformis group (BLg group). After the 72-h challenge, the Illumina-based paired-end sequencing approach to analyze the transcriptome profile of the crucian carp gut following AH infection. A total of 472.05 million valid reads were found from the four intestinal samples and 403.18 million reads were mapped successfully, of which 243.34 million were unique. About GO enrichment analysis, significant differentially expressed genes (DEGs) were more in biological process (406 DEGs), regulation of transcription, DNA-templated (345 DEGs), and signal transduction (304 DEGs). In the cell components, membrane (1913 DEGs) and integral component of membrane (1789 DEGs) had a higher number of significantly different genes. In the molecular function, metal ion binding (780 DEGs), ATP binding (535 DEGs), transferase activity (491 DEGs), and nucleotide binding (482 DEGs) have a large number of significantly different genes. Regarding the KEGG enrich analysis section, this study is arranged in ascending P-value order, andthe most significant signaling paths are lysosome, glucose metabolism, cycline cycline receiver interaction, and phagosome. Among them, the cycline cycline receiver interaction has the highest difference in significant genes. In addition, 5 immune-related genes were selected for real-time fluorescence quantitative PCR analysis to verify the RNA-seq data, and the results showed that the expression of all 5 genes was upregulated, which was consistent with the results of our RNA-seq analysis. After analysis, it was found that B. licheniformis can alleviate the inflammatory response and protect the fish body. Our results provide a reference for further analysis of the antagonism and mitigation mechanism of B. licheniformis against AH. [ABSTRACT FROM AUTHOR]

Aeromonas is the main pathogen causing bacterial diseases in fish. The disadvantages of chemical drugs to control fish diseases have been highlighted, and it is urgent to find an eco-friendly control method. In this study, an actinomycete strain with antibacterial activity against fish pathogenic bacteria was screened from soil samples. Combined with morphological characteristics, physiological and biochemical characteristics, and gyrB gene and whole genome comparison analysis, it was identified as a new strain of Streptomyces enissocaesilis, named Streptomyces enissocaesilis L-82. The strain has broad-spectrum antibacterial activity against fish pathogens. A substance with a mass-to-charge ratio of 227.20 [M + H] + was isolated and purified by high-performance liquid chromatography and mass spectrometry. It was presumed to be a derivative of 5-dimethylallylindole-3-acetonitrile. The strain is safe and non-toxic to crucian carp, and can stably colonize crucian carp and inhibit the proliferation of A. hydrophila. After feeding the feed containing 1 × 108 CFU/mL strain concentration, the weight growth rate and specific growth rate of crucian carp increased, the activity of ACP and SOD in serum increased, and the survival rate of crucian carp increased after challenge. Genome-wide analysis showed that the strain had strong ability to metabolize and tolerate extreme environments. And has a strong potential for disease resistance. Therefore, the strain is expected to be developed as a feed additive for fish farming. Key points: • The new Streptomyces enissocaesilis L-82 has a broad spectrum and stable antibacterial activity and meets the safety standards of feed additives. • Strain L-82 can colonize crucian carp, improve the growth, antioxidant, and immune performance of the host, and improve the survival rate after being infected with A. hydrophila. • Genome-wide analysis suggests that the strain has great disease resistance potential and is expected to be developed as a feed additive for fish culture. [ABSTRACT FROM AUTHOR]

The survival of farmed fish can be threatened by incidence of Aeromonas hydrophila infection. In addition, gut infection may promote dysbiosis of gut flora and dysregulate immune response in gut-liver axis in teleost fish. The current study aimed to evaluate the pathogenesis of A. hydrophila on gut barrier breach and synchronized immune response in gut-liver axis of RCCs. RCCs were subjected to continuous anal-intubation with A. hydrophila. Midgut sections were stained with periodic acid-Schiff (PAS) and immune-related gene expressions and biochemical indices in gut-liver axis were investigated. The severe midgut injury was detected in RCC anal-intubated with A. hydrophila, along with atrophy of the midgut villi, and the expression levels of tight junction (TJ) genes were consistently lower than those of the control (P < 0.05). Moreover, RCC receiving anal intubation with A. hydrophila showed the upregulation of pathological indices and immure genes in the midgut and liver. These findings demonstrated that gut infection with A. hydrophila can significantly disrupt epithelial permeability to exacerbate gut barrier breach and increase bacterial burdens in tissues as well as dysregulate immune response in gut-liver axis of RCC. [ABSTRACT FROM AUTHOR]

Aeromonas is the main pathogen causing bacterial diseases in fish. The disadvantages of chemical drugs to control fish diseases have been highlighted, and it is urgent to find an eco-friendly control method. In this study, an actinomycete strain with antibacterial activity against fish pathogenic bacteria was screened from soil samples. Combined with morphological characteristics, physiological and biochemical characteristics, and gyrB gene and whole genome comparison analysis, it was identified as a new strain of Streptomyces enissocaesilis, named Streptomyces enissocaesilis L-82. The strain has broad-spectrum antibacterial activity against fish pathogens. A substance with a mass-to-charge ratio of 227.20 [M + H] + was isolated and purified by high-performance liquid chromatography and mass spectrometry. It was presumed to be a derivative of 5-dimethylallylindole-3-acetonitrile. The strain is safe and non-toxic to crucian carp, and can stably colonize crucian carp and inhibit the proliferation of A. hydrophila. After feeding the feed containing 1 × 108 CFU/mL strain concentration, the weight growth rate and specific growth rate of crucian carp increased, the activity of ACP and SOD in serum increased, and the survival rate of crucian carp increased after challenge. Genome-wide analysis showed that the strain had strong ability to metabolize and tolerate extreme environments. And has a strong potential for disease resistance. Therefore, the strain is expected to be developed as a feed additive for fish farming. Key points: • The new Streptomyces enissocaesilis L-82 has a broad spectrum and stable antibacterial activity and meets the safety standards of feed additives. • Strain L-82 can colonize crucian carp, improve the growth, antioxidant, and immune performance of the host, and improve the survival rate after being infected with A. hydrophila. • Genome-wide analysis suggests that the strain has great disease resistance potential and is expected to be developed as a feed additive for fish culture. [ABSTRACT FROM AUTHOR]

In order to study the effect of adding Bacillus licheniformis to the diet on the liver and intestinal tissue structure, antioxidant capacity and immune function of crucian carp, a total of 360 healthy crucian carp selected for the experiment, which were divided into blank control group (CK group), Bacillus licheniformis test group (DY group), Aeromonas hydrophila attack group (CKG group) and Bacillus licheniformis treatment group (DYG group), CK group and CKG group were fed with basic diet, the DY and DYG groups added Bacillus licheniformis to the basic feed. Each group had three replicates, each with 30 crucian carp. After the 6-week feeding test, the CKG group and DYG group crucian carp were challenged by intraperitoneal injection of Aeromonas hydrophila to determine the cumulative death rate within 14 days. The results showed as follows: compared with CK group, DY group significantly increased the activity of acid phosphatase (ACP) and the contents of complement 3 (C3), complement 4 (C4) and immunoglobulin M (IgM) in serum of carp (P<0.05) and the activity of alkaline phosphatase (AKP) had an increasing trend but not significant. DY group significantly increased the activities of superoxide dismutase (SOD) and catalase (CAT) in serum and decreased the content of malondialdehyde (MDA) (P<0.05), after challenge, DYG group significantly inhibited oxidative damage and improved immune ability. Compared with the CK group, the length, width and muscular thickness of intestinal villus in DY group were significantly increased(P<0.05). After challenge, intestinal damage occurred in CKG group, intestinal villi were broken, intestinal epithelial cells were arranged disordered, striatum was arranged irregularly, and intestinal epithelial cells were vacuolated. Compared with CKG group, DYG group had less damage, striatum and epithelial cells were arranged neatly, and cell boundaries were obvious. The histological results of liver showed that there was no significant difference between the CK group and DY group. After the challenge, the liver nuclei were displaced and disordered, hepatocytes were damaged, nucleolysis occurred, vacuolation occurred, and hepatic sinus congestion occurred. Compared with CKG group, the liver cells in DYG group were better, the damage was reduced, the arrangement was gradually clear, and the vacuolation was relieved. The cumulative mortality of crucian carp in DYG group was significantly lower than CKG group(P<0.05). The addition of Bacillus licheniformis to the diet can not only effectively improve the antioxidant capacity of crucian carp serum, improve its immune function, but also increase the intestinal villus height and muscle layer thickness of crucian carp, and improve the damage of liver tissue structure. [ABSTRACT FROM AUTHOR]

MnSOD is a ubiquitous metalloenzyme that constitutes the first line of antioxidant defense against oxidative stress. In this study, full length sequence of MnSOD was identified from hybrid crucian carp (WR, Carassius cuvieri ♀ × Carassius auratus red var ♂). Tissue-specific analysis revealed that the highest expression of WR-MnSOD was detected in kidney. Aeromonas hydrophila challenge could dramatically enhance WR-MnSOD mRNA expression in tissues. In vivo administration of purified WR-MnSOD peptide could maintain gut mucosal barrier function, rescue redox balance as well as decrease apoptotic cell death in midgut upon bacterial infection, suggesting that WR-MnSOD is playing a crucial role in gut mucosal barrier function and could be used as feed additive to improve gut immunity in fish. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila can pose a great threat to the survival of farmed fish. In current study, we investigated the pathological characteristics and immune response in gut–liver axis of white crucian carp (WCC) upon gut infection. WCC anally intubated with A. hydrophila exerted a tissue deformation in damaged midgut with elevated levels of goblet cells along with a significant decrease in tight junction proteins and villi length‐to‐width ratios. In addition, immune‐related gene expressions and antioxidant properties increased dramatically in gut–liver axis of WCC following gut infection with A. hydrophila. These results highlighted the immune modulation and redox alteration in gut–liver axis of WCC in response to gut infection. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs), as the members of pattern-recognition receptors (PRRs), play a crucial role in innate immunity due to its significant function to recognize pathogens and activate downstream elements in signaling pathway. In the present study, the full-length cDNA sequences of TLR1 and TLR2 were cloned in the natural triploid Qihe crucian carp (Carassius auratus). The complete cDNA sequence of TLR1 was 2754 bp which was comprised of 5′ UTR of 60 bp, 3′ UTR of 300 bp, and an open reading frame of 2394 bp encoding 797 amino acids. The full-length cDNA sequence of TLR2 was 2605 bp, containing 5′ UTR of 36 bp, 3′ UTR of 202 bp, and an open reading frame of 2367 bp, encoding 788 amino acids. The mRNA expressions of CaTLR1 and CaTLR2 were detected to be constitutive at various embryonic developmental stages, and it was indicated that the similar expression pattern that mRNA expression levels were high at early development, then went down, and increased at later development stage. With regard to mRNA expression levels of CaTLR1 and CaTLR2 in various tissues of Qihe crucian carp, it showed to be constitutive with being higher in the liver and heart and the lowest in ovary. The mRNA expressions of CaTLR1 and CaTLR2 were profiled by quantitative real-time PCR following intraperitoneal injections of Aeromonas hydrophila and TLR ligands including LPS, PGN, and PolyI:C. Compared with the control, both CaTLR1 and CaTLR2 expressions were upregulated in most tissues of fish after challenges of LPS, PGN, PolyI:C, or A. hydrophila, although expression patterns varied in different tissues (gill, liver, spleen, trunk kidney, and head kidney). Together, these data highlighted the important role of CaTLR1 and CaTLR2 in recognizing LPS, PGN, PolyI:C, and A. hydrophila and in augmenting the innate immune ability against bacterial and viral infections in Qihe crucian carp. [ABSTRACT FROM AUTHOR]

Antibiotics were the main fishery drugs for treating Aeromonas hydrophila (A. hydrophila) infection, which would generate selective pressure and result in the appearance of antibiotic-resistant bacteria. The bacterial quorum sensing (QS) system provides a new alternative strategy against A. hydrophila infection. QS inhibitors can reduce bacterial virulence behaviors by disrupting QS, which has no effect on bacterial growth. Therefore, we studied the effect of cinnamaldehyde from a natural plant extract on the QS of A. hydrophila aiming to reduce its pathogenicity. The efficacy of cinnamaldehyde against A. hydrophila was evaluated from various aspects, including the effects on aerolysin, lipase, protease, swarming motility, biofilm formation, acyl-homoserine lactones (AHLs), and QS-related genes. Moreover, the therapeutic effect of cinnamaldehyde in vitro and in vivo was studied. The results showed that cinnamaldehyde could decrease the virulence phenotypes of A. hydrophila regulated by QS. Moreover, the transcriptions of related genes (aerA, ahyR, and ahyI) were downregulated following the addition of cinnamaldehyde. The in vitro and in vivo therapeutic assays show that cinnamaldehyde could reduce the aerolysin-mediated A549 cell injury and increase the survival rate of crucian carp infected with A. hydrophila. These results indicate that cinnamaldehyde would be a candidate QS inhibitor against A. hydrophila infection. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila , a Gram-negative bacterium, is one of the major pathogens causing bacterial sepsis in aquatic animals due to drug resistance and pathogenicity, which could cause high mortality and serious economic losses to the aquaculture. Sanguisorba officinalis (called DiYu in Chinese, DY) is well known as herbal medicine, which could inhibit the growth of pathogenic bacteria, hemostasis and regulate the immune response. Moreover, the active ingredients in DY could remarkably reduce drug resistance. In this study, we investigated the effects of probiotic fermentation cultures on A. hydrophila through in vitro and in vivo experiments. Three lactic acid bacteria, including Lactobacillus rhamnosus (LGG), Lactobacillus casei (LC) and Lactobacillus plantarum (LP), were selected to ferment the Chinese herbal medicine DY. The assays of antagonism showed that all three fermented cultures could influence the ability of A. hydrophila growth, among which L. rhamnosus fermented DY cultures appeared to be the strongest inhibitory effect. In addition, the biofilm determination revealed that L. rhamnosus fermented DY cultures could significantly inhibit the biofilm formation of A. hydrophila compared to the other groups. Furthermore, protease, lecithinase and urease activities were found in the three fermentation cultures. Three probiotics fermented DY cultures were orally administration with crucian carp to evaluate the growth performance, immunological parameters and pathogen resistance. The results showed that the three fermentation cultures could promote the growth performance of crucian carp, and the immunoglobulins, antioxidant-related enzymes and immune-related genes were significantly enhanced. Besides, the results showed that crucian carp received L. rhamnosus (60.87%), L. casei (56.09%) and L. plantarum (41.46%) fermented DY cultures had higher survival rates compared with the control group after infection with A. hydrophila. Meanwhile, the pathological tissue results revealed that the probiotic fermented cultures could largely improve the tissues damage caused by the pathogenic bacteria. In conclusion, this study proved that the fermentation cultures of three probiotics could effectively inhibit the growth of A. hydrophila , regulate the level of immune response and improve the survival rate against A. hydrophila in crucian carp. The present data suggest that probiotic fermented Sanguisorba officinalis act as a potential gut-targeted therapy regimens to protecting fish from pathogenic bacteria infection. The dietary supplementation of lactic acid bacteria fermented herbal Sanguisorba officinalis preparation for crucian carp (Carassius auratus) could improve the growth performance, antioxidant capacity, immune parameters and protection against Aeromonas hydrophila. [Display omitted] • Three lactic acid bacteria fermented herbal Sanguisorba officinalis (DY) cultures as dietary supplements for crucian carp. • The Chinese herbal fermented cultures improved the growth performance and non-specific immunity of crucian carp. • The Chinese herbal fermented cultures enhanced the resistance of crucian carp against A. hydrophila. [ABSTRACT FROM AUTHOR]

We evaluated the effect of dietary supplementation with Moringa oleifera leaf extract on the resistance to Aeromonas hydrophila infection in crucian carp. The fish were randomly divided into five groups: the basal diet, the basal diet supplied with 0.25% (0.25 M), 0.5% (0.5 M), 0.75% (0.75 M) and 1.0% M. oleifera leaf extract (1.0 M) for 8 weeks. The growth, antioxidant capabilities, related immune genes as well as resistance to A. hydrophila infection were determined. The results showed that compared with the control group, the weight gain, specific growth rate in the group of 0.5% M. oleifera leaf extract, serum superoxide dismutase (SOD), albumin (ALB) and glutathione peroxidase (GSH-Px), the gene expression of hepatopancreas BTB and CNC homolog 1 (Bach1), NF-E2-related factor 2 (Nrf2), peroxidases (PRX) and NADPH oxidase (NOX) in the group of 0.5%–1.0% M. oleifera leaf extract increased, while feed conversion ratio, serum cortisol, red blood cell (RBC), alanine aminotransferase (ALT), malonaldehyde (MDA) decreased in the group of 0.5%–1.0% M. oleifera leaf extract before the stress. After the infection, the group of 0.5% or 0.75% M. oleifera leaf extract also could improve the serum ALB, hepatopancreas Kelch-like-ECH-associated protein 1 (Keap1), Bach1 , Nrf2 , TOR , PRX and NOX and reduce cortisol compared with the control group. In summary, this study suggested that 0.5% M. oleifera leaf extract inclusion increased the growth performance, even had positive effects on physiological and immune function, and enhanced resistance against pathogenic infections in crucian carp. The optimum level of M. oleifera leaf extract for crucian carp was estimated to be 0.35%–0.48% based on polynomial comparison with FCR and SGR. • oleifera leaf extract has the potential to improve the growth of crucian carp. • oleifera leaf extract has the potential to improve resistance of bacteria pathogen. • oleifera leaf extract has the potential to immune and anti-oxidative ability. • oleifera leaf extract has the potential to up-regulate immune related genes of crucian carp. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila is a common pathogen of freshwater fish. In this study, A. hydrophila infection was shown to cause tissue damage, trigger physiological changes as well as alter the expression profiles of immune‐ and metabolic‐related genes in immune tissues of red crucian carp (RCC). Transcriptome analysis revealed that acute A. hydrophila infection exerted a profound effect on mitochondrial oxidative phosphorylation linking metabolic regulation to immune response. In addition, we further identified cellular senescence, apoptosis, necrosis and mitogen‐activated protein kinase signal pathways as crucial signal pathways in the kidney of RCC subjected to A. hydrophila infection. These findings may have important implications for understanding modulation of immunometabolic response to bacterial infection. [ABSTRACT FROM AUTHOR]

Fish skin is an essential protective barrier and functions as the first line of immune defense against pathogens. However, the molecular mechanism at the proteome-level remains unclear in the skin of fish. In this study, the comparative proteomics of skin immune responses of crucian carp Carassius auratus infected with Aeromonas hydrophila was investigated by isobaric tags for relative and absolute quantification (iTRAQ), two-dimensional gel electrophoresis combined with mass spectrometry (2-DE/MS) as well as high-throughput transcriptome (RNA-seq) techniques. A total of 241 and 178 differentially expressed proteins (DEPs) at 6 and 12 h post-infection (hpi) were respectively identified by iTRAQ, and key-DEPs were furtherly verified with 2-DE/MS analysis. GO and KEGG analysis showed that these DEPs were mostly related to metabolism, regulation of the cytoskeleton, stress and immune responses. Co-association results of proteome and transcriptome revealed the lysozyme (LYZ), complement C3, DnaJ (Hsp40) homolog subfamily C member 8 (DNAJC8) and allograft inflammatory factor 1-like (AIF1L) play important roles in skin immune responses of crucian carp. The significantly up-regulated expression of detected immune-related genes (c3 , mapk3 , f5, nlr, hsp90, itgb2, fnl, flnca, p47, mhc and pros1) were validated by qRT-PCR analysis. To our knowledge, this is first report on multi-omics analysis of the differential proteomics for the skin immune response of C. auratus against A.hydrophila infection, which contribute to the understanding the mechanisms of skin mucosal immunity in cyprinid fish. • Comparative proteome for skin immune response crucian carp were investigated by using by multi-omics analysis. • DEPs of skin were mostly related to metabolic pathways, regulation of the cytoskeleton, and stress and immune responses. • Lysozyme, complement, DnaJ (Hsp40) homolog and allograft inflammatory factor play important roles in skin immune response. • This is the first report on the molecular mechanisms of skin immunity in C. auratus against A. hydrophila at protein level. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila is a Gram-negative bacterial pathogen with a broad host range, including fish and humans. In this study, we examined the function of a secretory serine protease (named Ssp1) identified in pathogenic A. hydrophila CCL1. Ssp1 possesses a trypsin-like serine protease domain and contains two conserved PDZ domains. Recombinant Ssp1 protein (rSsp1) treatment increased intestinal permeability by downregulating and redistributing tight junction protein Occludin in intestinal Caco-2 cells in vitro. Western blot demonstrated that rSsp1 treatment in Caco-2 cells resulted in marked increases in the expressions of myosin light chain kinase (MLCK) and phosphorylated myosin light chain (p-MLC). For virulence analysis, an isogenic CCL1 mutant Δ Ssp1 was created. Δ Ssp1 bears an in-frame deletion of the Ssp1 gene. A live infection study in crucian carps showed that, compared to CCL1, Δ Ssp1 infection exhibited increased Occludin expression, reduced intestinal permeability and tissue dissemination capacity, and attenuated overall virulence in vivo. However, Δ Ssp1 showed no differences in the biofilm formation, swimming motility, and resistance to environmental stress. These lost virulence capacities of Δ Ssp1 were restored by complementation with the Ssp1 gene. Global transcriptome analysis and quantitative real-time RT-PCR showed that compared to CCL1 infection, Δ Ssp1 promoted the expressions of antimicrobial molecules (MUC2 , LEAP-2 , Hepcidin-1 , and IL-22). Finally, CCL1 infection caused significant dysbiosis of the gut microbiota, including increased Vibrio and Deefgea compared to Δ Ssp1 infected fish. Taken together, these results indicate that Ssp1 is essential for the virulence of A. hydrophila and is required for the perturbation of intestinal tight junction barrier. • A virulent Aeromonas hydrophila CCL1 was isolated from the diseased crucian carp. • rSsp1 increased intestinal permeability in intestinal Caco-2 cells in vitro. • Δ Ssp1 infection reduced intestinal permeability and attenuated overall virulence in vivo. [ABSTRACT FROM AUTHOR]

In mammals, β-catenin participates in innate immune process through interaction with NF-κB signaling pathway. However, its role in teleost immune processes remains largely unknown. We aimed to clarify the function of β-catenin in the natural defense mechanism of Qi river crucian carp (Carassius auratus). β-catenin exhibited a ubiquitous expression pattern in adult fish, as indicated by real-time PCR analysis. Following lipopolysaccharide (LPS), Polyinosinic-polycytidylic acid (polyI: C) and Aeromonas hydrophila (A. hydrophila) challenges, β-catenin increased in gill, intestine, liver and kidney, indicating that β-catenin likely plays a pivotal role in the immune response against pathogen infiltration. Inhibition of the β-catenin pathway using FH535, an inhibitor of Wnt/β-catenin pathway, resulting in pathological damage of the gill, intestine, liver and kidney, significant decrease of innate immune factors (C3 , defb3 , LYZ-C , INF-γ), upregulation of inflammatory factors (NF-κB , TNF-α , IL-1 , IL-8), and downregulation of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities, increase of Malondialdehyde (MDA) content. Following A. hydrophila invasion, the mortality rate in the FH535 treatment group exceeded that of the control group. In addition, the diversity of intestinal microflora decreased and the community structure was uneven after FH535 treatment. In summary, our findings strongly suggest that β-catenin plays a vital role in combating pathogen invasion and regulating intestinal flora in Qi river crucian carp. • β-catenin were upregulated in different tissues after pathogen challenge.. • Inhibition of β-catenin led to pathological damage of tissues.. • Inhibition of β-catenin resulted in low immune defense capacity. • Inhibition of β-catenin destroyed intestinal flora structure. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) have been recognized as safe microorganism that improve micro-flora disturbances and enhance immune response. A well-know traditional herbal medicine, Acanthopanax senticosus (As) was extensively utilized in aquaculture to improve growth performance and disease resistance. Particularly, the septicemia, skin wound and gastroenteritis caused by Aeromonas hydrophila threaten the health of aquatic animals and human. However, the effects of probiotic fermented with A. senticosus product on the immune regulation and pathogen prevention in fish remain unclear. Here, the aim of the present study was to elucidate whether the A. senticosus fermentation by Lactobacillus rhamnosus improve immune barrier function. The crucian carp were fed with basal diet supplemented with L. rhamnosus fermented A. senticosus cultures at 2 %, 4 %, 6 % and 8 % bacterial inoculum for 8 weeks. After trials, the weight gain rate (WGR), specific growth rate (SGR) were significantly increased, especially in LGG-6 group. The results confirmed that the level of the CAT, GSH-PX, SOD, lysozyme, and MDA was enhanced in fish received with probiotic fermented product. Moreover, the L. rhamnosus fermented A. senticosus cultures could trigger innate and adaptive immunity, including the up-regulation of the C3, C4, and IgM concentration. The results of qRT-PCR revealed that stronger mRNA transcription of IL-1β, IL-10, IFN-γ, TNF-α, and MyD88 genes in the liver, spleen, kidney, intestine and gills tissues of fish treated with probiotic fermented with A. senticosus product. After infected with A. hydrophila , the survival rate of the LGG-2 (40 %), LGG-4 (50 %), LGG-6 (60 %), LGG-8 (50 %) groups was higher than the control group. Meanwhile, the pathological damage of the liver, spleen, head-kidney, and intestine tissues of probiotic fermentation-fed fish could be alleviated after pathogen infection. Therefore, the present work indicated that L. rhamnosus fermented A. senticosus could be regard as a potential intestine-target therapy strategy to protecting fish from pathogenic bacteria infection. The dietary supplementation of different doses lactic acid bacteria fermented herbal Acanthopanax senticosus preparation for crucian carp (Carassius auratus) could improve the growth performance, antioxidant capacity, immune parameters and disease resistance against Aeromonas hydrophila. [Display omitted] • Different concentrations of L. rhamnosus fermented A. senticosus cultures were orally administration with crucian carp. • L. rhamnosus -fermented A. senticosus cultures improve the innate immune response in crucian carp. • L. rhamnosus fermented A. senticosus could up-regulate the level of IL-1β,IL-10,IFN-γ,TNF-α and MyD88 in various tissues. • L. rhamnosus fermented A. senticosus could be regard as a potential intestine-target therapy strategy. [ABSTRACT FROM AUTHOR]

Polymeric immunoglobulin receptor (pIgR) is an important member of the immunoglobulin superfamily. It can mediate polarity transportation of polymeric immunoglobulin in mucosal epithelial cells and play an important role in innate and acquired immunity. In this study, the full‐length cDNA sequence of Carassius auratus indigentiaus pIgR gene was firstly cloned and characterized. The complete cDNA sequence of C. auratus indigentiaus pIgR gene is 1399 bp, including a 984 bp open reading frame that encodes 327 amino acids. The pIgR is composed of extracellular, transmembrane and intracellular regions, which are composed of 255, 23 and 49 amino acids, respectively. The extracellular region contains two Ig‐like domains (ILDs), ILD1 consists of 104 amino acids (25–128) and ILD2 consists of 93 amino acids (137–229). The similarity between the pIgR sequences of C. auratus indigentiaus and other fish is 65.95%–97.55%. Analysis of phylogenetic trees shows that pIgR of C. auratus indigentiaus clusters into one branch with the pIgR of Cypriniformes. QRT‐PCR analysis revealed that the pIgR gene expression was highest in liver, followed by intestine, gill, head kidney and skin. After challenge with Aeromonas hydrophila, the relative expressions of pIgR in different tissues first increased and then decreased within 96 h. Moreover, the expression of pIgR gene peaked earlier in mucosal immune tissues (intestines, gill and skin) than in system immune tissues. It suggested that pIgR might play an important role in mucosa‐associated lymphoid tissue to protect the C. auratus indigentiaus against Aeromonas hydrophila infection. [ABSTRACT FROM AUTHOR]

A strain YJ4 with antibacterial activity was isolated and screened from the intestine of crucian carp, its fermentation broth has strong bacteriostatic effect on Aeromonas hydrophila, Vibrio harveii, Vibrio alginolyticus, Vibrio parahaemolyticus and Aeromonas verge. Morphological characteristics, physiological and biochemical characteristics, 16S rDNA sequence and phylogeny and phylogenetic evolution were analyzed. The results showed that strain YJ4 had 99% homology with Bacillus subtilis, which was identified as Bacillus subtilis. The property study found that Bacillus subtilis YJ4 bacteria liquid was resistant to acid, salt and high temperature, had no toxic effect on fish. The experiment indicates that Bacillus subtilis YJ4 isolated has inhibitory effect on common aquatic pathogens, which has strong tolerance. It is suitable for adding to aquatic feed to prepare pellets and can be used as aquatic microecological preparations. [ABSTRACT FROM AUTHOR]

Ladderlectin is a pattern recognition receptor (PRR) in fish that is critical for rapid detection of bacteria in vitro , but the immunological function of ladderlectin in vivo is essentially unknown. In this study, we examined the expression and function of a ladderlectin homologue (WR-ladderlectin) from hybrid crucian carp. WR-ladderlectin contains 157 amino acids and possesses the conserved C-type lectin domain. WR-ladderlectin is mainly expressed in the intestine and is upregulated by bacterial infection. Recombinant WR-ladderlectin (rWR-ladderlectin) agglutinated Aeromonas hydrophila and Escherichia coli. rWR-ladderlectin also bound the A. hydrophila and E. coli in a protein dose-dependent manner. As well as its ability to bind bacterial cells, rWR-ladderlectin displayed apparent bactericidal activity against A. hydrophila and E. coli in vitro. When introduced in vivo , rWR-ladderlectin induced significant expression of the antimicrobial molecules and tight junctions in the intestine. In addition, rWR-ladderlectin prevented significant decrease in the length of intestine villus and enhanced the host's resistance to bacterial infection. These results indicate that WR-ladderlectin is a classic pattern recognition molecule that protects intestinal mucosal barrier against bacterial infection. • mRNA expression of WR-ladderlectin was significantly induced by bacterial challenge. • WR-ladderlectin exhibited bactericidal activity to Aeromonas hydrophila and Escherichia coli. • WR-ladderlectin could enhance intestinal barrier and resistance against bacterial infection. [ABSTRACT FROM AUTHOR]

Aeromonas phage AHP-1 was originally isolated from crucian carp (Carassius carassius) tissue. It was able to infect Aeromonas hydrophila and A. salmonicida. Genome sequence analysis revealed a 218,317-bp-long linear genome with an overall G + C content of 47.9%, 315 open reading frames (ORFs), and 25 tRNA sequences. Its genome was found to contain 67 unique ORFs (21.26%) that did not show any homology to previously characterized proteins. A comparative genome analysis suggested that its closest neighbors are unclassified phages belonging to the genus Tequatrovirus of the subfamily Tevenvirinae. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila (A. hydrophila) is a typical zoonotic pathogenic bacterium that infects humans, animals, and fish. It has been reported that the Fur , a Fe2+ regulatory protein, and the Crp , a cAMP receptor protein, play important roles in bacterial virulence in many bacteria, but no research has been investigated on A. hydrophila. In this study, the Δ fur and Δ crp mutant strains were constructed by the suicide plasmid method. These two mutant strains exhibited a slightly diminished bacterial growth and also were observed some alterations in the number of outer membrane proteins, and the disappearance of hemolysis in the Δ crp strain. Animal experiments of crucian carp showed that the Δ fur and Δ crp mutant strains significantly decreased virulence compared to the wild-type strain, and both mutant strains were able to induce good immune responses by two kinds of administration routes of intraperitoneal immunization (i.p) and immersion immunization, and the protection rates through intraperitoneal injection of Δ fur and Δ crp to crucian carp were as high as 83.3 % and 73.3 %, respectively, and immersion immunization route of Δ fur and Δ crp to crucian carp provided protection as high as 40 % and 20 %, respectively. These two mutant strains showed abilities to induce changes in enzymatic activities of the non-specific enzymes SOD, LZM, AKP, and ACP in crucian carp. Together, these results indicated the Δ fur and Δ crp mutants were safe and effective candidate vaccine strains, showing good protection against the wild-type A. hydrophila challenge. • The genes of fur and crp are responsible for the virulence of Aeromonas hydrophila. • The fur and crp mutants induce good immunity by i.p and immersion immunization. • These two mutants provide good protection against the wild-type challenge of A. hydrophila in fish. [ABSTRACT FROM AUTHOR]

IL-22 is a critical cytokine of epithelial mucosal barrier. In humans, IL-22 signals through a heteroduplex receptor consisting of IL-22R and IL-10Rβ. In fish, IL-22 and its receptors homologues have been cloned in a number of species, however, no studies have been reported how the receptors are involved in IL-22 transduction. For this purpose, in this study we identified IL-22 and its soluble receptor IL-22BP and transmembrane receptors IL-22RA1 and IL-10R2 in Carassius cuvieri × Carassius auratus red var. (named WR-IL-22, WR-IL-22BP, WR-IL10R2 and WR-IL22RA1, respectively). WR-IL-22, WR-IL-22BP, WR-IL10R2 and WR-IL22RA1 were relatively conserved in the evolutionary process, sharing the same conserved domains as their higher vertebrate homologues. When the fish were infected with the Aeromonas hydrophila , the expression of WR-IL-22, WR-IL-22BP, WR-IL10R2 and WR-IL22RA1 were significantly induced in the gut. The co-IP assay showed that WR-IL-22 not only interacted with WR-IL-22BP, but also with WR-IL10R2 and WR-IL22RA1. When introduced in vivo , WR-IL-22 activated the JAK1-STAT3 axis and protected the gut mucosa from A. hydrophila infection. However, overexpression of WR-IL-22BP or knockdown of transmembrane receptors WR-IL10R2 and WR-IL22RA1 significantly inhibited the activation of WR-IL-22-mediated JAK1-STAT3 axis and promoted bacterial colonization in the gut. These results provided new insights into the role of IL-22 and its receptors in the gut mucosa barrier and immune response in teleost. • mRNA expression of WR-IL-22 was significantly induced by bacterial challenge. • WR-IL-22 interacted with WR-IL-22BP, WR-IL10R2 and WR-IL22RA1. • WR-IL-22 enhanced host gut barrier during Aeromonas hydrophila infection. [ABSTRACT FROM AUTHOR]

The experiment was to investigate the immune protective effect of Astragalus, Diyu, and Chinese gallnut on immune protection of crucian carp. 200 crucian carp were selected and randomly divided into five groups, 40 in each group. The control group was fed with a basic bait, and the experimental group was fed with bait coated with traditional Chinese medicine. The concentrations of Astragalus, Diyu, Chinese gallnut and combined treatment were 2.500, 2.500,1.250 and 0.625 g/mL, respectively. The traditional Chinese medicine decoction was mixed with the bait in a ratio of 1∶1,and the crucian carp was fed regularly and quantitatively. The test period was 50 d. The results showed that compared with control group, the content of serum immunoglobulin M（IgM） and the activity of lysozyme（LZM） in combination group were significantly increased（P<0.05）, and the content of complement C3 was extremely increased（P<0.01）. The activity of catalase（CAT） in the combination group was extremely increased（P<0.01）, and the activity of superoxide dismutase（SOD） was significantly increased（P<0.05）. The levels of interleukin-1β（IL-1β）, tumor necrosis factor-α（TNF-α）,interferon-γ（IFN-γ）, signaling pathway NF-KB, and MYD88 in the carp kidney and spleen in each experimental group all increased trend. Observation for 14 d after the challenge of Aeromonas hydrophila showed that traditional Chinese medicine could alleviate the infection of Aeromonas hydrophila on crucian carp, improve the survival rate of crucian carp,and play a protective role to a certain extent. The experiment indicates that the three traditional Chinese medicines can play a protective role in the immune protection of crucian carp. [ABSTRACT FROM AUTHOR]

FerL, a multifunctional iron-storage polypeptide, not only exhibited a regulatory role in iron metabolism, but also participated in the regulation of fish immunity. In this study, ORF sequence of WR-FerL was 522 bp, encoding 173 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-FerL was detected in spleen. A. hydrophila challenge and LPS stimulation could sharply enhance WR-FerL mRNA expression in tissues and fish cells, respectively. Purified WR-FerL fusion peptide exhibited in vitro binding activity to A. hydrophila and endotoxin, limited bacterial dissemination to tissues as well as attenuated A. hydrophila -induced production of pro-inflammatory cytokines. Moreover, WR-FerL overexpression could abrogate NF-κB and TNFα promoter activity in fish cells. These results indicated that WR-FerL could play an important role in host defense against A. hydrophila infection. • A novel FerL gene was characterized in hybrid crucian carp. • A. hydrophila challenge and LPS stimulation could increase expressions of WR-FerL mRNA. • WR-FerL could elicit a binding activity to A. hydrophila and LPS. [ABSTRACT FROM AUTHOR]

Ficus carica polysaccharides (FCPS), one of the most effective and important compo-nents in Ficus carica L., had been considered to be a beneficial immunostimulant and may be used in immunotherapy for animals and human. However, studies were little about the effect of FCPS used as immunomodulatory and the suitable dosage in fish. The present study investigated the effect of four different dietary levels of FCPS (0.1%, 0.2%, 0.4%, 0.8%) on the growth performance, innate immune responses and survival of crucian carp against Aeromonas hydrophila infection. The results showed that compared with control group, dietary FCPS had positive effects the growth performance (final weight, feed conversion ratio and survival rate) of crucian carp. FCPS induced significant higher (p < 0.05) leukocyte phagocytosis activity, serum bactericidal activity, lysozyme activity, com-plement C3, SOD activity and total protein level in the serum of crucian carp. Moreover, innate immune response of fish in FCPS groups increased first and then decreased with increasing dietary FCPS from 0.1% to 0.8%, and reached up to the peak in 0.4% dietary FCPS groups. Besides, the cumulative mortalities in FCPS groups were remarkably lower than that of control group when challenged with A. hydrophila , the relative percent survivals were 22.67%, 55.56%, 62.22% and 17.78% in 0.1% group, 0.2% group, 0.4% group and 0.8% group, respectively. These results suggested that dietary FCPS could improve the growth performance, innate immune response and disease resistance against A. hydrophila in fish, and the suitable dietary dose of FCPS was 0.4% in crucian carp. • Four different levels of FCPS all can improve the growth performance of crucian carp. • Four different levels of FCPS all enhance innate immune responses and survival of crucian carp against A. hydrophila infection. • The effect of different levels of FCPS on the innate immune response and survival was 0.4% FCPS >0.2% FCPS >0.1% FCPS >0.8% FCPS. • The suitable dietary dose of FCPS was 0.4% in crucian carp. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila can pose a great threat to survival of freshwater fish. In this study, A. hydrophila challenge could promote the erythrocyte hemolysis, increase free hemoglobin (FHB) level and generate malondialdehyde (MDA) production in plasma but decrease the levels of total antioxidant capacity (T-AOC), total superoxide dismutase (SOD), catalase (CAT), alkaline phosphatase (ALP) and lysozyme (LZM) of red crucian carp (RCC, 2 N = 100) and triploid hybrid fish (3 N fish, 3 N = 150) following A. hydrophila challenge. Elevated expression levels of heat shock protein 90 alpha (HSP90α), matrix metalloproteinase 9 (MMP-9), free fatty acid receptor 3 (FFAR3), paraoxonase 2 (PON2) and cytosolic phospholipase A2 (cPLA2) were observed in A. hydrophila- infected fish. In addition, A. hydrophila challenge could significantly increase expressions of cortisol, leucine, isoleucine, glutamate and polyunsaturated fatty acids (PUFAs) in RCC and 3 N, while glycolysis and tricarboxylic acid cycle appeared to be inactive. We identified differential fatty acid derivatives and their metabolic networks as crucial biomarkers from metabolic profiles of different ploidy cyprinid fish subjected to A. hydrophila infection. These results highlighted the comparative metabolic strategy of different ploidy cyprinid fish against bacterial infection. • Erythrocytes of triploid cyprinid fish exhibited a higher resistance against A. hydrophila -induced hemolysis. • Acute A. hydrophila infection could dramatically decrease antioxidant status in different ploidy cyprinid fish. • Acute A. hydrophila infection could trigger the difference in fatty acid derivatives in different ploidy cyprinid fish. [ABSTRACT FROM AUTHOR]

Galectin‐9, an important pathogen recognition receptor (PRR), could recognize and bind pathogen‐associated molecular patterns (PAMPs) on the surface of invading microorganisms, initiating the innate immune responses. A galectin‐9 was identified from Qihe crucian carp Carassius auratus and designated as CaGal‐9. The predicted CaGal‐9 protein contained two non‐identical carbohydrate recognition domains (CRDs), namely, N‐CRD and C‐CRD. The recombinant proteins (rCaGal‐9, rN‐CRD and rC‐CRD) were purified from Escherichia coli BL21 (DE3) and exhibited strong agglutinating activity with erythrocytes of rabbit. The haemagglutination was inhibited by D‐galactose, α‐lactose and N‐acetyl‐D‐galactose. Results of microbial agglutination assay showed that three recombinant proteins agglutinated Gram‐negative bacterium Aeromonas hydrophila and Gram‐positive bacterium Staphylococcus aureus. With regard to the binding activity, each recombinant protein could bind to LPS, PGN and the examined microorganisms (A. hydrophila and S. aureus) with different binding affinities. The integrated analyses suggested that CaGal‐9 with two CRD domains could play an important role in immune defence against pathogenic microorganisms for C. auratus. [ABSTRACT FROM AUTHOR]

NK-lysin, an effector of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), not only exhibits cytotoxic effect in fish cells, but also participates in the immune defense against pathogenic infection. In this study, ORF sequences of RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin were 369 bp. Tissue-specific analysis revealed that the highest expressions of RCC-NK-lysin and WCC-NK-lysin were observed in gill, while the peaked level of WR-NK-lysin mRNA was observed in spleen. A. hydrophila infection sharply increased RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin mRNA expression in liver, trunk kidney and spleen. In addition, elevated levels of NK-lysin mRNA were observed in cultured fin cell lines of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) after Lipopolysaccharide (LPS) challenge. RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin exerted regulatory roles in inducing ROS generation, modulating mitochondrial membrane potential, decreasing fish cell viability and antagonizing survival signalings, respectively. RCC/WCC/WR-NK-lysin-overexpressing fish could up-regulate expressions of inflammatory cytokines and decrease bacterial loads in spleen. These results indicated that NK-lysin in hybrid fish contained close sequence similarity to those of its parents, possessing the capacities of cytotoxicity and immune defense against bacterial infection. • Structure of NK-lysin in hybrid offspring was compared with those of its parents. • Up-regulated expressions of RCC/WCC/WR-NK-lysin were observed following A. hydrophila challenge and LPS stimulation. • RCC/WCC/WR-NK-lysin could cause the loss of MMP activity and decrease cell viability. [ABSTRACT FROM AUTHOR]

TNFα is one of important cytokines belonging to TNF superfamily, which can exhibit a pleiotropic effect in immune modulation, homeostasis as well as pathogenesis. However, its immunoregulatory function on mucosal immunity in fish gut are still unclear. In this study, we aimed to investigated the immunoregulatory role of TNFα1 in midgut of white crucian carp (WCC). WCC-TNFα1 sequence and its deduced structure were firstly identified in WCC. Then, tissue-specific analysis revealed that high-level WCC-TNFα1 expression was detected in gill. After Aeromonas hydrophila and lipopolysaccharide (LPS) stimulated, increased trends of WCC-TNFα1 expressions were detected in immune-related tissues and cultured fish cells, respectively. WCC anal-intubated with WCC-TNFα1 fusion protein showed the increased levels of edema and fuzzy appearance in impaired villi, along with atrophy and reduction of goblet cells (GC). Moreover, the expression levels of tight junction (TJ) genes and mucin genes were consistently lower than those of the control (P < 0.05). WCC-TNFα1 treatment could sharply decrease antioxidant status in midgut, while the expression levels of caspase (CASP) genes, unfolded protein response (UPR) genes and redox response genes increased dramatically. Our results suggested that WCC-TNFα1 could exhibit a detrimental effect on antioxidant and mucosal immune regulation in midgut of WCC. • The deduced structure and expression patterns of WCC-TNFα1 were firstly reported in WCC. • WCC receiving WCC-TNFα1 perfusion showed a dramatically reduced expressions of tight junction and mucin. • WCC-TNFα1 perfusion induced apoptotic activation but decreased antioxidant status in midgut [ABSTRACT FROM AUTHOR]

TNFα is a multifunctional cytokine that can modulate immune homeostasis and promote cellular regulation in mammals, but the crucial role of TNFα in fish gut remained unclear. The current study aimed to evaluate the immunoregulatory function of TNFα2 on gut barrier breach by A. hydrophila and systemic pathogenesis in gut and liver of WR (White crucian carp ♀ × red crucian carp ♂). WR-TNFα2 encoded 232 amino acid residues and showed a high-level of WR-TNFα2 expression in spleen. After A. hydrophila and lipopolysaccharide (LPS) stimulated, elevated levels of WR-TNFα2 expressions were detected in immune-related tissues and cultured fish cells, respectively. Gut perfusion with WR-TNFα2 alone could alter antioxidant status, increase extrinsic apoptosis as well as promote inflammatory response in midgut, causing severe injury of epithelial barrier. Moreover, WR-TNFα2-treated midgut exhibited a decline of mucosal protection against invasive A. hydrophila , which could elevate bacterial burden in tissues, aggravate tissue injury as well as attenuate immune activation. Our results suggested that WR-TNFα2 could exhibit a detrimental effect on gut barrier defense against A. hydrophila infection and dysregulate immune response in midgut and liver of hybrid crucian carp. • The predicted structure and expression patterns of TNFα2 were firstly reported in WR. • WR-TNFα2 altered antioxidant status and increased extrinsic apoptosis in midgut. • WR-TNFα2 facilitated bacterial infection by impairing gut barrier and then exacerbated liver lesion. [ABSTRACT FROM AUTHOR]

Some medicinal plants have been known as immunostimulants, and the medicinal plants extract has been used to control the outbreak of the disease in aquaculture for many years. In this study, a total of 270 crucian carp (30 ± 5 g) were randomly distributed in 9 aquaria (55 cm l × 40 cm w × 50 cm h) and divided into three feeding groups including 0 (Control), 50 mg kg−1 (Diet A) and 100 mg kg−1 (Diet B) of salidroside. The expression of immune-related genes (IL-1β, TNFα, MYD88, CXCL-8, TGF-β, and IL-11) in the kidney had a significant increase when the crucian carp fed with Diet B for 4 weeks (P < 0.05). Meanwhile, the expression of IL-1β, TNFα, and CXCL-8 in the spleen was significantly up-regulated when the fish fed with Diet B (P < 0.05). Higher serum alkaline phosphatase (AKP) activity, catalase (CAT) activity, superoxide dismutase (SOD) activity, and complement C3 content were found in the fish which fed with salidroside-supplemented diet. Our results also proved that fish fed with salidroside-supplemented diet for four weeks, especially at a concentration of 100 mg kg−1 diet, improved the protection of crucian carp against A. hydrophila. The amount of A. hydrophila in the kidney and spleen was significantly decreased in salidroside-supplemented diet groups (P < 0.05). In conclusion, the present results demonstrate that the addition of salidroside for four weeks can improve the immune response of crucian carp and increase the protection against the pathogen, especially at the concentration of 100 mg kg−1 diet. The protective effect of the salidroside to the crucian carp could be used as alternatives to antibiotics for controlling fish diseases in aquaculture. • Salidroside evaluated as an immunostimulant in crucian carp. • Salidroside has strong immunostimulant effects up-regulated immune related gene expression. • Salidroside has strong immunostimulant effects stimulated serum enzyme activity. • The use of salidroside in crucian carp diet was increased fish resistance against A. hydrophila infection. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are non-coding RNA molecules that regulate gene expression in fish, but its regulatory mechanism of the skin mucosal immune response remains poorly understood. In order to investigate the immunological role of miRNAs, three sRNA libraries (mSC, mST1, mST2) from skin samples of crucian carp (Carassius auratus) infected with Aeromonas hydrophila at three time points (0, 6 and 12 hpi) were constructed and examined using Illumina Hiseq 2000 platform. All of the identified miRNA, rRNA and tRNA were 69444 (13.39%), 29550 (5.70%) and 10704 (2.06%) in skin, respectively. At 6 and 12 hpi, 829 and 856 miRNAs were differentially expressed, respectively. Among these DEMs, 53 known and 10 novel miRNAs were all significantly differentially expressed during early infection (p < 0.01). GO and KEGG enrichment analyses revealed that 118111 target-genes were primarily involved in cellular process, metabolic process, biological regulation and stress response, such as antigen processing and presentation, complement and coagulation cascades, phagosome, MAPK, TLR, NF-κB and JAK-STAT signaling pathways. These results will help to elucidate the mechanism of miRNAs involved in the skin mucosal immune response of crucian carp against Aeromonas hydrophila infection. • This was first report on skin miRNAs profiles of C. auratus upon A. hydrophila infection. • 829 and 856 DEMs in skin were identified at 6 and 12 hpi, respectively. • 10 novel miRNAs were significantly differentially expressed during early infection. • Antigen presentation, complement, MAPK, TLR and NF-κB pathways were significantly involved in skin mucosal immunity. [ABSTRACT FROM AUTHOR]

Galectin-9, as one of the important PRRs in host, could initiate the immune defense responses through recognizing and binding PAMPs on the surface of invading microorganisms. In this study, a new galectin-9 cDNA was identified and characterized in Qihe crucian carp Carassius auratus (named as CaGal-9). The complete cDNA sequence of CaGal-9 was 1318 bp, with an open reading frame (ORF) of 963 bp encoding 320 amino acids. The predicted CaGal-9 protein contained two non-identical carbohydrate recognition domains (CRDs), which possessed the representative motifs H-NPR and WG-EER to bind with β-galactoside. Based on the RT-qPCR detection, CaGal-9 was ubiquitously expressed at mRNA level in various tested tissues, and predominately expressed in spleen. Upon Aeromonas hydrophila and poly I: C challenge, the expressions of CaGal-9 were remarkably up-regulated in liver, spleen, kidney and head kidney in a time-depended manner. The recombinant CaGal-9 (rCaGal-9), purified from Escherichia coli BL21 (DE3), exhibited strong binding ability with lipopolysaccharide (LPS), peptidoglycan (PGN) and β-Glucan, as well as the examined microorganisms including fungus, Gram-negative bacteria, and Gram-positive bacteria. With regard to the agglutinating activity of rCaGal-9, it could agglutinate erythrocytes of rabbit and crucian carp, and the examined microorganisms. Taken together, in this study, it was suggested that CaGal-9 could play an important role in immune defense against pathogenic microorganisms in C. auratus , which functions as an important PRR to recognize PAMPs and agglutinate pathogenic microorganisms. • A novel galectin-9 CaGal-9 was characterized in Carassius auratus. • CaGal-9 mRNA expressions were obviously up-regulated after pathogen challenges. • Recombinant CaGal-9 exhibited binding ability with LPS, PGN and β-Glucan. • Recombinant CaGal-9 could agglutinate erythrocytes and microorganism. • Recombinant CaGal-9 exhibited binding ability with seven kinds of microorganism. [ABSTRACT FROM AUTHOR]

Lipocalin 2 (Lcn2) has been identified in mammals, however, the in vivo function of fish Lcn2 is essentially unknown. Triploid crucian carp (3n = 150) of red crucian carp (female, 2n = 100) and allotetraploid (male, 4n = 200) shows better resistance to pathogenic infections. To elucidate the antimicrobial mechanism of triploid crucian carp, we examined the function of a novel Lcn2 from triploid crucian carp (3nLcn2). 3nLcn2 is 183 residues in length and contains a conserved lipocalin domain. Quantitative real time reverse transcription PCR (qRT-PCR) analysis showed that 3nLcn2 expression occurred in multiple tissues and was upregulated by bacterial infection in a time-dependent manner. We found that purified recombinant 3nLcn2 (r3nLcn2) exerted bactericidal activity to Aeromonas hydrophila and Escherichia coli. qRT-PCR detected increased expression of pro-inflammatory cytokines and tight junctions in fish with 3nLcn2 overexpression. Fish administered with 3nLcn2 exhibited enhanced intestinal barrier and resistance against bacterial infection. These results provide the first evidence that 3nLcn2 is a functional lipocalin with antimicrobial activity and plays a positive role in the immune defense during bacterial infection. • mRNA expression of 3nLcn2 was significantly induced by bacterial challenge. • 3nLcn2 exhibited bactericidal activity to Aeromonas hydrophila and Escherichia coli. • 3nLcn2 could increase the expression of pro-inflammatory cytokines and tight junctions. • 3nLcn2 could enhance intestinal barrier and resistance against bacterial infection. [ABSTRACT FROM AUTHOR]

Hepcidin, a multifunctional hormone oligopeptide, not only exhibits a regulatory role in iron metabolism, but also participates in the regulation of teleostean immunity. In this study, ORF sequence of WR-hepcidin was 258 bp and encoded 85 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-hepcidin was observed in liver. Aeromonas hydrophila challenge can sharply increased WR-hepcidin mRNA expression in liver, trunk kidney and spleen. The purified WR-hepcidin fusion peptide can directly bind to A. hydrophila and Streptococcus agalactiae , reduce the relative bacterial activity, limit bacterial growth and attenuate their dissemination to tissues in vivo. In addition, the treatment of WR-hepcidin fusion protein can diminish the production of pro-inflammatory cytokines. These results indicated that WR-hepcidin can play a negative regulatory role in bacteria-stimulated pro-inflammatory cytokines production and MyD88-IRAK4 activation. • We characterized a novel hepcidin gene in hybrid crucian carp. • The up-regulation of WR-hepcidin was observed following bacterial challenge. • WR-hepcidin can bind to gram positive and gram negative bacteria. • WR-hepcidin can restrict the bacterial growth and reduce bacteria-stimulated inflammatory response. [ABSTRACT FROM AUTHOR]

Myostatin, through type I receptor (kinase 4, 5, ALK4/5), functions to participate in the immune system and negatively regulate muscle growth in mammals. However, the role of myostatin (mstn) in the immune system of teleosts is largely unknown. In a previous study, we cloned the mstn1 cDNA encoding myostatin in Qi river crucian carp (Carassius auratus). In the present study, we have cloned mstn2 cDNA, which was characterized and analyzed together with mstn1. Tissue distribution analysis showed that both mstn genes are expressed in numerous tissues, with mstn1 dominantly expressed in the muscle and brain, whereas mstn2 is mainly expressed in the brain. During embryogenesis, mstn1 and mstn2 exhibit different expression patterns. Both mstn1 and mstn2 expression increased stepwise in the brain at different developmental stages. Furthermore, both genes are differentially regulated during different periods of fasting/re-feeding. Following the exposure of C. auratus to polyI:C, lipopolysaccharide (LPS), and Aeromonas hydrophila , both genes were upregulated in different tissues, which indicated that they might be involved in the immune response against pathogenic invasion. Blocking the Mstn signal pathway with SB-431542 (a chemical inhibitor of ALK4/5) resulted in significantly increased body length and weight. However, the mortality of SB-431542-treated fish was higher after A. hydrophila challenge. Moreover, decreased expression of lysozymes (lyz), complement component 3 (c3), β-defensin 3 (defb3), and interferon γ (ifnγ) were exhibited in treated fish, compared with the controls. Furthermore, the expression of nf-κb1 , three pro-inflammatory cytokines (il1 β , il6, and tnfα), and inflammatory cytokines (il8 and il10) were significantly increased in both the SB-431542-treated group and the control after A. hydrophila infection, suggesting that the NF-κB pathway was not suppressed in the SB-431542-treated fish. Taken together, our data suggest that both mstn1 and mstn2 play important roles in early body development, muscle growth, and the immune system by acting downstream of the NF-κB signal pathway. • Mstn1 and mstn2 exhibit different tissue distribution patterns. • Both mstn genes increased stepwise in the brain at different developmental stages. • Two mstn genes were upregulated in different tissues after pathogen challenge. • Inhibition of Mstn signal pathway resulted in larger body size, but low immune defense capacity. [ABSTRACT FROM AUTHOR]

Skin plays an important role in the innate immune responses of fish, particularly towards bacterial infection. To understand the molecular mechanism of mucosal immunity of fish during bacterial challenge, a de novo transcriptome assembly of crucian carp Carassius auratus skin upon Aeromonas hydrophila infection was performed, the latter with Illumina Hiseq 2000 platform. A total of 118111 unigenes were generated and of these, 9693 and 8580 genes were differentially expressed at 6 and 12 h post-infection, respectively. The validity of the transcriptome results of eleven representative genes was verified by quantitative real-time PCR (qRT-PCR) analysis. A comparison with the transcriptome profiling of zebrafish skin to A. hydrophila with regards to the mucosal immune responses revealed similarities in the complement system, chemokines, heat shock proteins and the acute-phase response. GO and KEGG enrichment pathway analyses displayed the significant immune responses included TLR, MAPK, JAK-STAT, phagosome and three infection-related pathways (ie., Salmonella , Vibrio cholerae and pathogenic Escherichia coli) in skin. To our knowledge, this study is the first to describe the transcriptome analysis of C. auratus skin during A. hydrophila infection. The outcome of this study contributed to the understanding of the mucosal defense mechanisms in cyprinid species. • First report on transcriptome in the skin of C. auratus against A. hydrophila infection. • Several unknown function genes were differentially expressed in the skin. • Twenty-eight significant immune pathways were involved in skin immune responses. • Comparative transcriptome analysis of zebrafish skin revealed several similarities. [ABSTRACT FROM AUTHOR]

Hepcidin is a small peptide of defensins with antibacterial activity, and plays an important role in innate immunity against pathogenic microorganisms, which can also participate in the regulation of iron metabolism. The hepcidin gene in Yellow River carp (Cyprinus carpio haematopterus) (CcHep) was cloned and identified. The total length of CcHep cDNA was 480 bp, containing an open reading frame (ORF) that encoded 91 amino acids (aa), which contained a 24-aa signal peptide, a 42-aa propeptide, and a 25-aa mature peptide. The mature peptide had a typical RX (K/R) R motif and eight conserved cysteine residues forming four pairs of disulfide bonds. Homology and phylogenetic tree analysis showed that CcHep had the closest relationship with that of crucian carp. The expression levels of hepcidin mRNA in healthy and Aeromonas hydrophila stimulated fish were measured by real-time fluorescence quantitative PCR. The results showed that CcHep mRNA was expressed in different tissues of healthy fish with the highest relative expression level in liver, followed by kidney and intestine, and the lowest expression level was observed in heart. The hepcidin gene was extremely significantly up-regulated in head kidney, intestine, liver, skin, spleen, and gill at 6 h and 12 h after A. hydrophila infection. Furthermore, the immunoregulation effect of dietary recombinant protein was evaluated. The recombinant hepcidin protein (rCcHep) was successfully expressed by Pichia pastoris X-33 and showed strong antibacterial activity against A. hydrophila , Escherichia coli , Vibrio anguillarum and Bacillus subtilis in vitro. In order to evaluate the preventive effect of rCcHep, fish were fed with basal diet or diet supplemented with different doses of rCcHep, and then challenged with A. hydrophila. The results showed that immune genes were up-regulated to varying degrees, and feed additive groups exhibited a significantly improved up-regulation expressions of Lysozyme , Toll-like receptor 5 (TLR 5), Major histocompatibility complex classⅡ (MHCⅡ), while inhibited up-regulation expressions of Interleukin 1β (IL-1β), Interleukin 8 (IL-8), and Tumor necrosis factor α (TNF-α) in liver and spleen compared to the control. Meanwhile, the relative immune protection rate in 120 mg/kg feed additive group was 28%, and the bacterial clearance rate in tissues of this group was higher than that of the control. Collectively, these results indicated that rCcHep had antibacterial activity and showed an immune protection effect against A. hydrophila , and could be considered as a dietary supplement to apply in aquaculture. • The hepcidin gene was cloned and gene characterization was studied in Yellow River carp. • Recombinant hepcidin protein showed strong antibacterial activity against bacteria in vitro. • Dietary recombinant protein showed protective effect on Yellow River carp challenged with A. hydrophila. [ABSTRACT FROM AUTHOR]

Abstract Myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6) are two critical signal transducers in toll-like receptor (TLR) pathway. In the present study, we identified and characterized the homologues of MyD88 and TRAF6 in Qihe crucian carp Carassius auratus , termed as CaMyD88 and CaTRAF6 , respectively, and examined their roles during pathogenic infection. Full-length cDNA of CaMyD88 was 2463 bp, including a 191 bp 5′-untranslated region (UTR), a 1417 bp 3′-UTR, and an 855 bp open reading frame (ORF) encoding for a putative protein with 284 amino acids. Full-length cDNA of CaTRAF6 was identified to be 2555 bp, consisting of a 52 bp 5′-UTR, an 871 bp 3′-UTR, and a 1632 bp ORF encoding a protein of 543 amino acids. Deduced amino acid sequences of CaMyD88 and CaTRAF6 contained the typical domains (CaMyD88: death domain and TIR domain; CaTRAF6: one RING-type zinc finger domain, two TRAF-type zinc finger domains, one coiled-coil region, and one conserved C-terminal meprin and TRAF homology domain) as in other fish. Quantitative Real-Time PCR (qRT-PCR) analysis revealed that both CaMyD88 and CaTRAF6 were ubiquitously expressed throughout the development stages and appeared to be developmentally regulated. In addition, CaMyD88 and CaTRAF6 had a broadly distribution of expression in all examined eleven tissues of healthy fish, although the transcript levels varied among the different tissues. Moreover, it was found that mRNA expressions of CaMyD88 and CaTRAF6 were generally up-regulated after stimulation by polyI:C, flagellin, and Aeromonas hydrophila in spite of the down-regulation appeared at some time points or tissues. These results indicated that CaMyD88 and CaTRAF6 play the critical roles in the immune defense of Qihe crucian carp against pathogenic invasion. The present findings will provide the valuable information for understanding the innate immune responses of Qihe crucian carp and contribute to develop the preventive way against pathogens. Highlights • Full length DNA and cDNA of MyD88 and TRAF6 were respectively characterized in Carassius auratus. • Maternal CaMyD88 or CaTRAF6 mRNA was gradually consumed in early period of developmental embryos. • Target gene mRNA expressions show obvious tissue specificity. • Expression patterns of two genes show the significant responses to immune challenges. [ABSTRACT FROM AUTHOR]

Abstract This study explored the immunomodulatory effect and inhibition effects of the candidate probiotic Lactococcus lactis 16-7, which was isolated from crucian carp, on Aeromonas hydrophila infection in crucian carp. The experimental fish were divided into two groups; one was fed a diet supplemented with L. lactis , while the other was fed the control probiotic-free diet. After feeding for 42 d with the experimental diets, the fish that received the diet supplemented with probiotics exhibited a significantly enhanced serum superoxide dismutase activity, phagocytic activities of innate immune cells, and the expression levels of immune-related genes [interferon-γ (INF-γ), interleukin-1β (IL-1β), interleukin-11 (IL-11), tumour necrosis factor α (TNF-α) and myeloid differentiation factor 88 (MyD88)], indicating that L. lactis 16-7 could activate the non-specific immune system of crucian carp. At the end of the feeding trial, the crucian carps in each group were orally infected with A. hydrophila NJ-35. The results show that L. lactis 16-7 could prevent the increase in d -lactic acid concentration and inflammatory response caused by A. hydrophila in crucian carp. Compared with A. hydrophila group, L. lactis 16-7 preserved the integrity of intestinal villi and mitigated A. hydrophila -induced reduce in the transcriptional levels of tight junction (TJ) proteins zonula occludens-1 (ZO-1) and occludin, indicating that L. lactis 16-7 could reduce intestinal mucosal barrier damage and inflammation induced by A. hydrophila in crucian carp. In addition, L. lactis 16-7 could effectively antagonize the colonization of A. hydrophila in the intestine. Overall, these data clearly indicate that L. lactis 16-7 has the potential to be developed as a probiotic agent against A. hydrophila infection in aquaculture. Highlights • Lactococcus lactis 16-7 could activate the non-specific immune system of crucian carp. • L. lactis 16-7 could reduce A. hydrophila -induced intestinal mucosal barrier function damage and inflammation. • L. lactis 16-7 could antagonize the colonization of A. hydrophila in the intestine. [ABSTRACT FROM AUTHOR]

The pharmacokinetic (PK) properties of enrofloxacin (ENR) and its metabolite ciprofloxacin (CIP) were investigated in crucian carp following oral administration at different dose levels (5, 10, 20, 40 mg/kg body weight). The disposition kinetics of ENR was found to be linear over the dose range studied. Serum half‐lives ranged from 64.56 to 72.68 hr. The in vitro and ex vivo activities of ENR in serum against a pathogenic strain of Aeromonas hydrophila were determined. In vitro and ex vivo bactericidal activity of ENR was concentration dependent. Dosing of 10 mg/kg resulted in an AUC/minimum inhibitory concentration (MIC) ratio of 368.92 hr and a Cmax/MIC ratio of 7.23, respectively, against A. hydrophila 147 (MIC = 0.48 μg/ml), indicating a likely high level of effectiveness in clinical infections caused by A. hydrophila with MIC value ≤ 0.48 μg/ml. Modeling of ex vivo growth inhibition data to the sigmoid Emax equation provided the values of AUC24 hr/MIC required to produce bacteriostasis, bactericidal activity, and elimination of bacteria, these values were 21.70, 53.01, and 125.21 hr, respectively. These findings in conjunction with MIC90 data suggested that ENR at the dose of 7.81 mg/kg predicted a positive clinical outcome and minimize the risk of emergence of resistance. [ABSTRACT FROM AUTHOR]

Cetobacterium somerae is a commensal bacterium for many fish species. However, research on C. somerae has been limited so far, and its function and beneficial potential require to be further investigated. The objective of this study was to evaluate the probiotic properties of C. somerae CPU-CS01 isolated from the intestinal contents of crucian carp (Carassius auratus). Hemolytic activity, antibiotic susceptibility, acid tolerance, bile salt tolerance, free radical scavenging, and enzyme production properties were tested for in vitro. Caenorhabditis elegans and zebrafish (Danio rerio) model were used to evaluate the antioxidant and anti-infective effects of C. somerae CPU-CS01 in vivo. Our results showed that C. somerae CPU-CS01 had no hemolytic activity, it produced cellulase, amylase, and survived at low pH (2.0–3.0) and in the presence of bile salts. The cell-free culture supernatant (CFCS) of C. somerae CPU-CS01 possessed DPPH radical, hydroxyl radical, and superoxide anion scavenging activity. C. elegans fed with C. somerae CPU-CS01 were more resistant to hydrogen peroxide-induced oxidative stress and Aeromonas hydrophila infection. In addition, zebrafish-fed diets containing C. somerae CPU-CS01 showed improved survival after A. hydrophila infection. Based on these results, the positive probiotic properties of C. somerae CPU-CS01 isolated from the intestinal contents of crucian carp make it a potential candidate for probiotic. • somerae CPU-CS01 was screened for probiotic potential. • CFCS of C. somerae CPU-CS01 possessed antioxidant activity. • somerae CPU-CS01 improved survival rate against A. hydrophila infection in C. elegans and zebrafish model. • somerae CPU-CS01 may be an intervention therapy strategy against bacterial infection. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila (A. hydrophila) , a gram-negative bacterium, causes serious diseases with various clinical symptoms in farm raised fish. Thus, different ways to prevent and control A. hydrophila infection need to be explored, including a vaccine. In this study, we evaluated the protective efficacy of an oral vaccine prepared from the A. hydrophila TPS maltoporin (Malt) with Lactobacillus plantarum (L. plantarum) against A. hydrophila infection in crucian carp (Carassius auratus). For the in vivo experiment, the oral vaccine was administered to crucian carp by feeding them fish diets containing Lp-pPG-Malt, Lp-pPG and PBS for 28 days. The enzyme-linked immunosorbent assay (ELISA), leukocyte phagocytosis assay and real-time quantitative polymerase chain reaction (RT-qPCR) were performed to measure the protective efficacy of the Lp-pPG-Malt. ELISA and leukocyte phagocytosis assay confirmed that Lp-pPG-Malt significantly enhanced the IgM level and nonspecific immune response of crucian carp compared with the control groups (Lp-pPG and PBS). The RT-qPCR results showed that the Lp-pPG-Malt increased the relative expression of immune-related genes (IL-10, IL-1β, TNF-α, IFN-γ) of crucian carp in various tissues (liver, spleen, head kidney and hind intestine). Moreover, Lp-pPG-Malt significantly increased the relative percent survival of fish after intraperitoneal injection with A. hydrophila (55%) compared with the Lp-pPG and PBS groups (0%). These findings suggest that Lp-pPG-Malt can serve as an oral vaccine candidate for A. hydrophila infection and that Malt can be used as an effective antigen in crucian carp farming. • A recombinant L. plantarum Lp-pPG-Malt as an oral vaccine candidate was conducted. • Efficacy of the Lp-pPG-Malt against A. hydrophila infection were evaluated in crucian carp. • The Lp-pPG-Malt can serve as a potent oral vaccine against A. hydrophila infection in crucian carp farming. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila can pose a great threat to fish survival. In this study, we investigated the differential immune and redox response in gut-liver axis of hybrid fish (WR) undergoing gut infection. WR anally intubated with A. hydrophila showed severe midgut injury with decreased length-to-width ratios of villi along with GC hyperplasia and enhanced antioxidant activities, but expression profiles of cytokines, chemokines, antibacterial molecules, redox sensors and tight junction proteins decreased dramatically. In contrast, immune-related gene expressions and antioxidant activities increased significantly in liver of WR following gut infection with A. hydrophila. These results highlighted the differential immune regulation and redox balance in gut-liver axis response to bacterial infection. [Display omitted] • Severe midgut injury with GC hyperplasia was observed in WR anally intubated with A. hydrophila. • Suppressions of tight junction molecules were observed in midgut of WR following gut infection. • WR receiving gut infection with A. hydrophila exhibited differential immune and redox response in gut-liver axis. [ABSTRACT FROM AUTHOR]

The collectin subfamily member 11 (Colec11), plays an important role in innate immunity as a pattern recognition molecule. In the present study, a colec11 homolog was identified and characterised from Qihe crucian carp, namely, Ca-colec11. The full-length cDNA of Ca-colec11 was composed of 1129 bp, with a 99 bp 5′-untranslated region (UTR), 816 bp open reading frame (ORF) encoding a 271-aa protein and 214 bp 3′-UTR with a polyadenylation signal sequence (aataaa) and a poly(A) tail. The deduced amino acid sequence of Ca-Colec11 contained a si gnal peptide, collagen domain, neck region and carbohydrate-recognition domain (CRD), which had four conserved cysteine residues (Cys170-Cys256 and Cys242-Cys264) and an EPN/WND motif required for carbohydrate-binding specificity. Tissue expression profile analysis by quantitative real-time polymerase chain reaction (RT-qPCR) showed that Ca-colec11 was ubiquitously distributed in the tested tissues and highly expressed in the liver. The gene expression levels of Ca-colec11 were evidently up-regulated in the liver, spleen, kidney and head kidney after infection with A. hydrophila and S. aureus. The recombinant Ca-Colec11 (rCa-Colec11) purified from Escherichia coli BL21 (DE3) could agglutinate A. hydrophila and S. aureus , and it possessed haemagglutination activity against rabbit erythrocytes, which was inhibited by various carbohydrates, including d -Mannose, N-Acetyl- d -mannosamine, l -Fucose, d -Glucose, N-Acetyl- d -glucosamine, d -Galactose, LPS and PGN. Furthermore, rCa-Colec11 could inhibit the growth of A. hydrophila and S. aureus. These findings collectively demonstrated that Ca-Colec11, as a PRR, could play a role in the immune defence of Qihe crucian carp. • Ca-colec11 was identified from Qihe crucian carp. • Ca-colec11 expression was obviously up-regulated after bacteria challenge. • rCa-Colec11 agglutinated erythrocytes, which was inhibited by various carbohydrates. • rCa-Colec11 agglutinated Aeromonas hydrophila and Staphylococcus aureus. • rCa-Colec11 inhibited the growth of Aeromonas hydrophila and Staphylococcus aureus. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila is one of important pathogenic bacteria that can imperil fish survival. Current study suggested that hybrid crucian carp (WR) receiving acute A. hydrophila challenge exerted a molecular regulation linking peroxisomal fatty acid β-oxidation to immune response. A. hydrophila infection may alleviate antioxidant status along with the significant alternation of immune-, apoptosis- and metabolic-related gene expressions in kidney, whereas most portions of differential metabolites (DMs) were enriched in steroid derivatives and phospholipids. In addition, mevalonate (MVA) pathway and phospholipid hydrolysis were identified as crucial cellular processes in WR after A. hydrophila challenge. These results may provide an insight into immunometabolic strategy against A. hydrophila infection. • Acute A. hydrophila infection could induce antioxidant imbalance in WR. • Changes of immune- and metabolic-related gene expressions in kidney were observed. • A. hydrophila infection exhibit a profound effect on MVA pathway and phospholipid hydrolysis. [ABSTRACT FROM AUTHOR]

Dexamethasone, a known immunosuppressant, can inhibit the immune response and increase the amount of pathogen in body, but the role of dexamethasone affecting susceptibility of crucian carp ( Carassius auratus ) to pathogen is unclear. The effects of dexamethasone on susceptibility of crucian carp to Aeromonas hydrophila were investigated in this study. The fish were divided into four groups randomly and injected intraperitoneally by dexamethasone for 0 day (group D), 3 days (group C), 6 days (group B), and 9 days (group A), respectively. The serum lysozyme activity was significantly declined in group A, B and C. Relative immune gene expression such as il - 1β , cxcl-8 , tnfα and crp in kidney were down-regulation compared to group D. After that crucian carp were infected with A. hydrophila , crucian carp treated by dexamethasone had higher mortality (group A 95%, group B 76%, group C 31%) when compared to group D (4% mortality); the amount of pathogen in was significantly increased ( P < 0.05) in liver, kidney and spleen of fish in group A-C compared to group D. These results implicated that higher susceptibility caused by dexamethasone may be induced by the decrease of lysozyme activity and the down-regulation of some immune genes. [ABSTRACT FROM AUTHOR]

The comparative pharmacokinetics of enrofloxacin ( ENR) and its metabolite ciprofloxacin ( CIP) were investigated in healthy and Aeromonas hydrophila-infected crucian carp after a single oral (p.o.) administration at a dose of 10 mg/kg at 25 °C. The plasma concentrations of ENR and of CIP were determined by HPLC. Pharmacokinetic parameters were calculated based on mean ENR concentrations by noncompartmental modeling. In healthy fish, the elimination half-life ( T1/2 λz), maximum plasma concentration ( Cmax), time to peak ( Tmax), and area under the concentration-time curve ( AUC) values were 64.66 h, 3.55 μg/ mL, 0.5 h, and 163.04 μg·h/ mL, respectively. In infected carp, by contrast, the corresponding values were 73.70 h, 2.66 μg/ mL, 0.75 h, and 137.43 μg·h/ mL, and the absorption and elimination of ENR were slower following oral administration. Very low levels of CIP were detected, which indicates a low extent of deethylation of ENR in crucian carp. [ABSTRACT FROM AUTHOR]

In this study, a total of 420 healthy crucian carp (9.77 ± 0.04 g) were randomly divided into CK, B·S , XOS and B·S + XOS group, and cultured for 8 weeks. Results showed that the dietary Bacillus subtilis (B. subtilis) and xylo-oligosaccharides (XOS) can significantly increased the final weight, weight gain, specific growth rate, feed efficiency, protein efficiency and survival rate of crucian carp. Dietary B. subtilis and XOS can significantly increased the activities of catalase, glutathione, superoxide dismutase and total antioxidant capacity, significantly decreased the contents of malondialdehyde, and significantly increased the activities of alkaline phosphatase, acid phosphatase, lysozyme and the contents of complement component 3,4 and immunoglobulin M in crucian carp serum. In addition, compared with CK group, the expression levels of TGF-β and IL-10 in B·S , XOS and B·S + XOS group were significantly increased, and the expression levels of TNF-α, HSP90, IL-1β, TLR4 and MyD88 were significantly decreased. Supplementation of B. subtilis and XOS can also improve the intestinal tissue morphology of crucian carp. After injection of 1 × 107 CFU/mL Aeromonas hydrophila (A. hydrophila), compared with CK group, the survival rates of the B·S group, the XOS group and the B·S + XOS group were increased by 13.98%, 10.56% and 30.74%, respectively. These results show that dietary B. subtilis and XOS can significantly improve the growth performance, antioxidant capacity, immunity and resistance to A. hydrophila of crucian carp, and the combined effect is better than that of single addition. The graphical abstract of CBC 109296 (Title: Boosted growth performance, immunity, antioxidant capacity and disease resistance of crucian carp (Carassius auratus) by single or in combination dietary Bacillus subtilis and xylo-oligosaccharides). [Display omitted] • Dietary B. subtilis and XOS can improve the growth performance, immunity and antioxidant capacity of crucian carp. • Supplementation of B. subtilis and XOS in feed can significantly improve the intestinal tissue morphology of crucian carp. • Dietary B. subtilis and XOS can significantly improve the resistance of crucian carp to Aeromonas hydrophila infection. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila can threaten the survival of freshwater fish. In this study, A. hydrophila challenge could induce tissue damage, promote antioxidant imbalance as well as alter the transcript levels of oxidative stress indicators, apoptotic genes and metabolic enzyme genes in kidney of red crucian carp (RCC). Metabolomics analysis revealed that A. hydrophila challenge had a profound effect on amino acid metabolism and lipid metabolism. In addition, we further identified dipeptides, fatty acid derivatives, cortisol, choline and tetrahydrocortisone as crucial biomarkers in kidney of RCC subjected to A. hydrophila infection. These results highlighted the importance of metabolic strategy against bacterial infection. [Display omitted] • Acute A. hydrophila infection could induce tissue damage in kidney of RCC. • Decreased level of antioxidant status was observed in kidney of RCC following A. hydrophila challenge. • hydrophila infection could decrease levels of dipeptides and fatty acid derivatives in kidney of RCC. [ABSTRACT FROM AUTHOR]