Feeding strategy and feed protein level affect the gut microbiota of common carp (Cyprinus carpio).

Common carp (Cyprinus carpio) were fed food with different protein concentrations following different feeding regimes, which were previously shown to affect growth, nitrogen excretion and amino acid catabolism. 16S rRNA gene amplicon sequencing was performed to investigate the gut microbiota of these fish. Lower dietary protein content increased microbial richness, while the combination of demand feeding and dietary protein content affected the composition of the gut microbiota. Hepatic glutamate dehydrogenase (GDH) activity was correlated to the composition of the gut microbiota in all dietary treatments. We found that demand-fed carp fed a diet containing 39% protein had a significantly higher abundance of Beijerinckiaceae compared to other dietary groups. Network analysis identified this family and two Rhizobiales families as hubs in the microbial association network. In demand-fed carp, the microbial association network had significantly fewer connections than in batch-fed carp. In contrast to the large effects of the feeding regime and protein content of the food on growth and nitrogen metabolism, it had only limited effects on gut microbiota composition. However, correlations between gut microbiota composition and liver GDH activity showed that host physiology and gut microbiota are connected, which warrants functional studies into the role of the gut microbiota in fish physiology.

Acanthopanax senticosus cultures fermented by Lactobacillus rhamnosus enhanced immune response through improvement of antioxidant activity and inflammation in crucian carp (Carassius auratus).

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-1ß, 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.

Isolation, Characterization, and Effect on Biofilm Formation of Bacteriocin Produced by Lactococcus lactis F01 Isolated from Cyprinus carpio and Application for Biopreservation of Fish Sausage.

The aim of this work was the screening of bacteriocin-producing LABs isolated from fish, the selection of promising/prominent strain(s), the characterization of the bacteriocin produced, and the evaluation of its potential to be used as biopreservative(s). Amplification and sequencing of the 16S rRNA gene of the bacteriocin-producing strain was performed. Then a partial purification of the produced bacteriocin, using a combination of ammonium sulfate and chloroform-methanol precipitation, was done. Its molecular weight was determined by SDS-PAGE. In addition, the action spectrum, the hemolysis test, and its ability to inhibit biofilm formation were analyzed. A total of 88 isolates of lactic acid bacteria (LAB) including one bacteriocin producer, which was identified as Lactococcus lactis F01, were collected. The bacteriocin was partially purified with an estimated yield of 40%. Regarding the SDS-PAGE profile, the secreted bacteriocin has molecular weight of about 3.5 kDa and was identified as class I bacteriocin. The antimicrobial test showed that the bacteriocin inhibits pathogenic and/or spoilage bacteria, 10 Gram-positive and 16 Gram-negative bacterial species. Moreover, it can inhibit biofilm formation from 1.3% (Escherichia coli) to 63.92% (Pseudomonas aeruginosa ATCC15692) depending on the strain. The hemolytic activity of novel bacteriocin was observed at the concentration of 10 µg/ml of bacteriocin crude extract, which was 0.7 ± 0.0029%. In addition, it exhibited good thermal and pH stability with retained antibacterial activity of 85.25% after treatment at 121°C for 20 min, as well as at a pH range between 2.0 and 10.0. Moreover, this bacteriocin showed the ability to inhibit the growth of bacterial culture load in fish sausage stored at 8°C for 28 days. Considering the results obtained, bacteriocin could be potentially exploited as an alternative to chemical preservatives or as a substitute for antibiotics.

Characterization of potential probiotic bacteria Enterococcus faecium MC-5 isolated from the gut content of Cyprinus carpio specularis.

The goal of this study was to determine the unique characteristics of Enterococcus faecium MC-5, a probiotic bacteria isolated from the intestine of a fish, Cyprinus carpio specularis, collected from Dal Lake in Srinagar, Kashmir, India. For this, the important valuable probiotic attributes, some functional properties, and safety assessments were analyzed in-vitro for the strain MC-5. The strain E. faecium MC-5 exhibited high resistance to low pH, high bile salt, lysozyme, and phenol. The strain MC-5 showed excellent auto- and co-aggregation properties and displayed remarkable hydrophobicity towards various tested hydrocarbons which suggested that the strain possesses venerable adhesion properties. Apart from these, the cell-free supernatant (CFS) of strain MC-5 exhibited phenomenal antimicrobial activity against the tested pathogens. A scanning electron microscope (SEM) image revealed strain MC-5 finely adhered to human colon adenocarcinoma cells (HCT-15 cells). The strain MC-5 showed high bile salt hydrolase activity and excellent cholesterol removal ability of 70.27%. The intact cells of strain MC-5 also showed strong DPPH scavenging activity. The EPS produced by E. faecium MC-5 inhibited the adhesion of Listeria monocytogenes, Staphylococcus aureus, and Salmonella enterica on HCT-15 cells with maximum inhibition rates of 41.82, 40.34, and 55.51%, respectively for displacement assay, which was higher as compared to exclusion (26.06, 26.11, and 39.23%) and competition assays (30.06, 26.7, and 41.20%). Strain MC-5 did not exhibit hemolysis and was also found susceptible to vancomycin and other clinically important antibiotics. When evaluating all the results from the present study, it is propounded that strain MC-5 has enviable probiotic characteristics and thus can be used as bio-protective cultures and/or bio-shield in food and pharmaceutical industries.

Heterogeneity of the Tissue-specific Mucosal Microbiome of Normal Grass Carp (Ctenopharyngodon idella).

Microbiome plays key roles in the digestion, metabolism, and immunity of the grass carp (Ctenopharyngodon idella). Here, we characterized the normal microbiome of the intestinal contents (IC), skin mucus (SM), oral mucosa (OM), and gill mucosa (GM) of the grass carp, as well as the microbiome of the sidewall (SW) of the raising pool, using full-length 16S rRNA sequencing based on the PacBio platform in this specie for the first time. Twenty phyla, 38 classes, 130 families, 219 genera, and 291 species were classified. One hundred four common classified species might be core microbiota of grass carp. Proteobacteria, Bacteroides, and Cyanobacteria were the dominant phyla in the niche of grass carp. Proteobacteria and Bacteroides dominated the taxonomic composition in the SM, GM, and OM, while Proteobacteria, Planctomycetota, and Cyanobacteria preponderated in the IC and SW groups. Microbiota of IC exhibited higher alpha diversity indices. The microbial communities clustered either in SW or the niche from grass carp, significantly tighter in the SW, based on Bray-Curtis distances (P < 0.05). SM, GM, and OM were similar in microbial composition but were significantly different from IC and SW, while IC had similarity with SW due to their common Cyanobacteria (P < 0.05). Differences were also reflected by niche-specific and differentially abundant microorganisms such as Noviherbaspirillum in the SM and Rhodopseudomonas palustris, Mycobacterium fortuitum, and Acinetobacter schindleri in GM. Significantly raised gene expression was found in IC and SM associated with cell cycle control, cell division, chromosome, coenzyme transport and metabolism, replication, recombination and repair, cell motility, post-translational modification, signal transduction mechanisms, intracellular trafficking, secretion, and vesicles by PICRUSt. This work may be of great value for understanding of fish-microbial co-workshops, especially in different niche of grass carp.

Selenium-enriched Bacillus subtilis reduces the effects of mercury-induced on inflammation and intestinal microbes in carp (Cyprinus carpio var. specularis).

Mercury (Hg) is a global pollutant that affects the health of humans and ecosystems. Selenium (Se) is an essential trace element for many organisms including humans. Bacillus subtilis is one of the main probiotics used in aquaculture, and has a certain adsorption effect on heavy metals. The interaction between Hg and Se was rigorously studied, especially due to the observation of the protective effect of Se on Hg toxicity. The objective of this study was to research the effects of Hg, Se, and B. subtilis on inflammation and intestinal microbes in common carp. The common carp was exposed to Hg (0.03 mg/L), and 105 cfu/g Se-rich B. subtilis was added to the feed. After 30 days of feeding, samples were taken to evaluate the growth performance, serological response, inflammatory response, and intestinal microbial changes. In this study, when fish were exposed to Hg, the growth performance of the Se-rich B. subtilis plus 0.03 mg/L Hg fish group was lower than that of the control group and higher than 0.03 mg/L Hg; The levels of serum immunoglobulin M (IgM) and lysozyme (LZM) decreased, but after supplementation with Se-rich B. subtilis, the levels of LZM and IgM increased; Hg treatment significantly upregulated the mRNA expression of interleukin-1ß (IL-1ß), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and nuclear factor-kB (NF-κB P65), but downregulated the mRNA expression of interleukin-10 (IL-10), transforming growth factor-ß (TGF-ß) and NF-kappa-B inhibitor alpha (IkBα). However, compared with the Hg group, the Se-rich B. subtilis plus Hg group can significantly increase the mRNA expression levels of IL-1ß, IL-8, TNF-α, and NF-κB P65, but reduce the regulation of IL-10, TGF-ß, and IkBα expression. Through the analysis of the microbiological, we found that the Hg group was mainly composed of Aeromonas sobria and Aeromonas hydrophila. However, in the Se-rich B. subtilis treatment group, we found that Aeromonas sobria was significantly less than the Hg group. Se-rich B. subtilis improves Hg-induced intestinal microbial changes, alleviates the abundance of Aeromonas, and alleviates the inflammation of the fish. The results of this study show that Se-rich B. subtilis dietary supplements can effectively protect common carp against Hg toxicity.

Bactericidal efficacy of non-thermal plasma activation against Aeromonas hydrophila and immunological responses of koi (Cyprinus carpio haematopterus).

In the aquaculture industry, an efficient and safe water purification system is important to prevent mass mortality by virulent pathogens. As extensive use of traditional methods (e.g.: povidone-iodine, ozone, ultraviolet irradiation, formalin, and chlorine dioxide) have adverse effects on cultured fish, an appropriate and alternative water purification method is vital for the sustainability of the industry. Non-thermal plasma technology has been successfully used for various biomedical purposes (e.g: food sterilization, medical device disinfection, wound healing, cancer therapy, etc.) and has great potential to be used as a sterilizing system. However, few studies have been conducted on its usefulness in the aquaculture industry. In this study, we investigated the bactericidal efficacy of plasma-activated water induced by non-thermal plasma and its histopathological as well as immunological adverse effects on koi. A highly virulent Aeromonas hydrophila SNU HS7, which caused massive mortality of koi, was used for this study. Non-thermal plasma was applied for 10 min to the fish tanks with 1.2 × 109 CFU/mL SNU HS7 using PLMB-20 system to confirm the sterilization efficacy and to observe the survival and immunological reaction of koi for 14 days. As a result, gross pathological, histopathological, and immunological investigations did not reveal any significant adverse effects in fish as compared to the control groups. To the best of our knowledge, this is the first study showing that non-thermal plasma can be used for sterilization of rearing water without giving significant physiological damage to the fish, even under the assumption of extreme situations. As plasma can effectively sterilize not only bacteria but also other unknown pathogens, the results of this study are showing a promising future in purifying water in aquaculture practice.

Vacuum impregnation of chitosan coating combined with water-soluble polyphenol extracts on sensory, physical state, microbiota composition and quality of refrigerated grass carp slices.

Herein, the effects of chitosan (CH) coating with different water-soluble polyphenol extracts (pomegranate peel (PPE), grape seed (GSE) and green tea (GTE)) through vacuum impregnation on the quality retention and microflora of refrigerated grass carp fillets were studied. Generally, the quality degradation of carp fillets was remarkably alleviated using coatings when compared to the control. As suggested by microbial enumeration and high-throughput sequencing, protective coatings were conductive to inhibit bacteria growth, especially spoilage bacteria of Pseudomonas. As a result, the indicator related to bacteria such as total volatile basic nitrogen (TVB-N) and K value had lower levels in coating groups than that in control. In addition, coating also slowed down the deterioration of physical properties of color, texture and water holding capacity in fillets, giving fillets a better edible quality. By contrast, the fillets treated by composite coatings had better quality during storage when compared to chitosan coating alone, and a relatively good synergistic antibacterial effect between chitosan and extracts was also observed, especially for CH-GTE. Overall, the best performance to inhibit quality deterioration was recorded in CH-GTE, with the lowest values of TVB-N, TBARS, K-value and water loss, and highest values of shear force and sensory preference among groups.

Dynamic Interaction Between Mucosal Immunity and Microbiota Drives Nose and Pharynx Homeostasis of Common Carp (Cyprinus carpio) After SVCV Infection.

The crosstalk between the immune system and microbiota drives an amazingly complex mutualistic symbiosis. In mammals, the upper respiratory tract acts as a gateway for pathogen invasion, and the dynamic interaction between microbiota and mucosal immunity on its surface can effectively prevent disease development. However, the relationship between virus-mediated mucosal immune responses and microbes in lower vertebrates remains uncharacterized. In this study, we successfully constructed an infection model by intraperitoneally injecting common carp (Cyprinus carpio) with spring viremia of carp virus (SVCV). In addition to the detection of the SVCV in the nose and pharynx of common carp, we also identified obvious histopathological changes following viral infection. Moreover, numerous immune-related genes were significantly upregulated in the nose and pharynx at the peak of SVCV infection, after which the expression levels decreased to levels similar to those of the control group. Transcriptome sequencing results revealed that pathways associated with bacterial infection in the Toll-like receptor pathway and the Nod-like receptor pathway were activated in addition to the virus-related Rig-I-like receptor pathway after SVCV infection, suggesting that viral infection may be followed by opportunistic bacterial infection in these mucosal tissues. Using 16S rRNA gene sequencing, we further identified an upward trend in pathogenic bacteria on the mucosal surface of the nose and pharynx 4 days after SVCV infection, after which these tissues eventually reached new homeostasis. Taken together, our results suggest that the dynamic interaction between mucosal immunity and microbiota promotes the host to a new ecological state.

Cyanobacteria Microcystis aeruginosa Contributes to the Severity of Fish Diseases: A Study on Spring Viraemia of Carp.

Fish are exposed to numerous stressors in the environment including pollution, bacterial and viral agents, and toxic substances. Our study with common carps leveraged an integrated approach (i.e., histology, biochemical and hematological measurements, and analytical chemistry) to understand how cyanobacteria interfere with the impact of a model viral agent, Carp sprivivirus (SVCV), on fish. In addition to the specific effects of a single stressor (SVCV or cyanobacteria), the combination of both stressors worsens markers related to the immune system and liver health. Solely combined exposure resulted in the rise in the production of immunoglobulins, changes in glucose and cholesterol levels, and an elevated marker of impaired liver, alanine aminotransferase (ALT). Analytical determination of the cyanobacterial toxin microcystin-LR (MC-LR) and its structurally similar congener MC-RR and their conjugates showed that SVCV affects neither the levels of MC in the liver nor the detoxification capacity of the liver. MC-LR and MC-RR were depurated from liver mostly in the form of cysteine conjugates (MC-LR-Cys, MC-RR-Cys) in comparison to glutathione conjugates (LR-GSH, RR-GSH). Our study brought new evidence that cyanobacteria worsen the effect of viral agents. Such inclusion of multiple stressor concept helps us to understand how and to what extent the relevant environmental stressors co-influence the health of the fish population.

2D-DIGE proteomic analysis of blood plasma reveals changes in immune- and stress-associated proteins following hormonal stimulation of carp males.

In carp aquaculture, hormonal manipulation with an analog of GnRH (Ovopel) and carp pituitary extract (CPE), which act at different levels of the hypothalamic-pituitary-gonadal axis, is a routine practice to enhance sperm production. Our recent studies revealed that hormonal stimulation of male carp was associated with changes in the seminal plasma proteome, including blood origin proteins. Here, we explored whether Ovopel and CPE could affect the blood proteome of male carp. Both preparations induced increases in semen volume, total number of sperm, and testosterone level. However, hormonal stimulation did not affect the plasma cortisol and glucose levels. A comparative proteomic analysis of carp blood plasma between the control (PBS) and the hormonally treated males revealed significant changes (>1.2 <-1.2-fold change, P < 0.05) in the abundance of 30 spots (14 up- and 16 downregulated) and 44 spots (28 up- and 16 downregulated) upon CPE and Ovopel treatment, respectively. The most significantly affected pathways were acute phase response signaling, the coagulation system, LXR/RXR and FXR/RXR activation; however, there were different sets of proteins in Ovopel- and CPE-treated males. The majority of differentially abundant proteins were involved in the regulation of the immune defense response, the response to stress, and complement activation. Moreover hormonal stimulation with CPE markedly increased the bactericidal activity of blood and both preparations caused profound changes in gene expression in hematopoietic organs. This work is important in understanding the biological processes behind the protein-based response to hormonal stimulation of sperm production in fish.

Ferritin H can counteract inflammatory response in hybrid fish and its parental species after Aeromonas hydrophila infection.

Ferritin H can participate in the regulation of fish immunity. Tissue-specific analysis revealed that the highest expressions of Ferritin H in parental species were observed in spleen, while peaked level of Ferritin H mRNA in hybrid fish was observed in liver. In addition, A. hydrophila challenge could sharply enhance their Ferritin H mRNA expression in liver, kidney and spleen. To further investigate their roles in immune regulation, their Ferritin H fusion proteins were produced in vitro. Ferritin H fusion proteins could exhibit a direct binding activity to A. hydrophila and endotoxin in a dose-dependent manner, restrict dissemination of A. hydrophila to tissues and abrogate inflammatory cascades. Moreover, treatment with Ferritin H fusion proteins could reduce A. hydrophila-induced lipid peroxidation. These results indicated that Ferritin H in hybrid fish elicited a similar immune regulation of A. hydrophila-induced inflammatory signals in comparison with those of its parents.

Functional miR-142a-3p Induces Apoptosis and Macrophage Polarization by Targeting tnfaip2 and glut3 in Grass Carp (Ctenopharyngodon idella).

In the process of microbial invasion, the inflammation reaction is induced to eliminate the pathogen. However, un-controlled or un-resolved inflammation can lead to tissue damage and death of the host. MicroRNAs (miRNAs) are the signaling regulators that prevent the uncontrolled progress of an inflammatory response. Our previous work strongly indicated that miR-142a-3p is related to the immune regulation in grass carp. In the present study, we found that the expression of miR-142a-3p was down-regulated after infection by Aeromonas hydrophila. tnfaip2 and glut3 were confirmed as be the target genes of miR-142a-3p, which were confirmed by expression correlation analysis, gene overexpression, and dual luciferase reporter assay. The miR-142a-3p can reduce cell viability and stimulate cell apoptosis by targeting tnfaip2 and glut3. In addition, miR-142a-3p also regulates macrophage polarization induced by A. hydrophila. Our results suggest that miR-142a-3p has multiple functions in host antibacterial immune response. Our research provides further understanding of the molecular mechanisms between miRNAs and their target genes, and provides a new insights for the development of pro-resolution strategies for the treatment of complex inflammatory diseases in fish.

Novel technique for treating grass carp (Ctenopharyngodon idella) by combining plasma functionalized liquids and Ultrasound: Effects on bacterial inactivation and quality attributes.

A novel technique for treating grass carp by combining plasma functionalized liquids and ultrasound to inactivate bacteria was developed. The effects of the plasma functionalized liquids (PFL) including plasma functionalized water (PFW) and buffer (PFB) and their respective combination with ultrasound treatment (USPFW and USPFB) on the oxidative and physical qualities of grass carp were also investigated. Individual applications of PFW and PFB significantly reduced the populations of Escherichia coli and Shewanella putrefaciens in the range of 0.31-1.18 log CFU/g, compared with the control with a reduction of 0.18 log CFU/g, while combined treatments of USPFW and USPFB presented additional reductions of 0.05-0.65 log CFU/g, with potential synergy demonstrated for PFW and ultrasound. The treatment resulted in improved biomedical index and nutritional value of fatty acids and lipids, protein structural unfolding, increased lipid oxidation and protein degradation with values within the acceptable limits, and the combined treatment was more effective for retarding the hardness reduction in grass carp, while the colour change was also significantly affected, resulting in increased whiteness. The results indicated that the combined treatments may be a promising approach to improving the quality of seafood products.

The immune response of pIgR and Ig to Flavobacterium columnare in grass carp (Ctenopharyngodon idellus).

The polymeric immunoglobulin receptor (pIgR) plays an important role in mediating the transcytosis of polymeric immunoglobulins (pIgs) to protect organisms against pathogen invasion. Here, a polyclonal antibody against grass carp (Ctenopharyngodon idellus) recombinant pIgR was developed by immunizing New Zealand white rabbit, and the responses of pIgR, IgM and IgZ were analyzed after bath immunization and intraperitoneal administration with Flavobacterium columnare. The results showed that pIgR transcription level was similar to IgM and IgZ, but pIgR rose much faster and peaked earlier than IgM and IgZ; the pIgR mRNA levels were higher in the skin and spleen for both immunized groups, while IgM and IgZ mRNA expression were higher in skin, gills, and intestines in bath immersion group, or spleen and head kidney in intraperitoneal immunization group. ELISA revealed that the IgM, IgZ and pIgR protein levels were up-regulated in skin mucus, gill mucus, gut mucus and bile, reaching a higher peak level earlier in skin mucus and gill mucus in bath immersion group, but a higher peak level in bile in injection group. Moreover, secretory component molecules were detected in grass carp's skin, gill and intestine mucus and bile, but not in serum, which molecular mass was near the theoretical mass obtained from the sequence of grass carp pIgR. These results demonstrated that bath and intraperitoneal immunization up-regulated pIgR and secretory Ig expression in secretions, which provided more insights into the role of pIgR in immunity and offer insight into ways of protecting teleost against pathogen invasion.

Evaluation of Alpha-Ketoglutarate Supplementation on the Improvement of Intestinal Antioxidant Capacity and Immune Response in Songpu Mirror Carp (Cyprinus carpio) After Infection With Aeromonas hydrophila.

As an intermediate substance of the tricarboxylic acid cycle and a precursor substance of glutamic acid synthesis, the effect of alpha-ketoglutarate on growth and protein synthesis has been extensively studied. However, its prevention and treatment of pathogenic bacteria and its mechanism have not yet been noticed. To evaluate the effects of alpha-ketoglutarate on intestinal antioxidant capacity and immune response of Songpu mirror carp, a total of 360 fish with an average initial weight of 6.54 ± 0.08 g were fed diets containing alpha-ketoglutarate with 1% for 8 weeks. At the end of the feeding trial, the fish were challenged with Aeromonas hydrophila for 2 weeks. The results indicated that alpha-ketoglutarate supplementation significantly increased the survival rate of carp after infection with Aeromonas hydrophila (P < 0.05), and the contents of immune digestion enzymes including lysozyme, alkaline phosphatase and the concentration of complement C4 were markedly enhanced after alpha-ketoglutarate supplementation (P < 0.05). Also, appropriate alpha-ketoglutarate increased the activities of total antioxidant capacity and catalase and prevented the up-regulation in the mRNA expression levels of pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1ß, interleukin-6, and interleukin-8 (P < 0.05). Furthermore, the mRNA expression levels of toll-like receptor 4 (TLR4), and nuclear factor kappa-B (NF-κB) were strikingly increased after infection with Aeromonas hydrophila (P < 0.05), while the TLR4 was strikingly decreased with alpha-ketoglutarate supplementation (P < 0.05). Moreover, the mRNA expression levels of tight junctions including claudin-1, claudin-3, claudin-7, claudin-11 and myosin light chain kinases (MLCK) were upregulated after alpha-ketoglutarate supplementation (P < 0.05). In summary, the appropriate alpha-ketoglutarate supplementation could increase survival rate, strengthen the intestinal enzyme immunosuppressive activities, antioxidant capacities and alleviate the intestinal inflammation, thereby promoting the intestinal immune responses and barrier functions of Songpu mirror carp via activating TLR4/MyD88/NF-κB and MLCK signaling pathways after infection with Aeromonas hydrophila.

Evaluation of probiotic properties of Lysinibacillus macroides under in vitro conditions and culture of Cyprinus carpio on growth parameters.

Cyprinus carpio is an important freshwater fish in aquaculture. It was used for the isolation of potential probiotic strain for aquaculture applications. The most dominant strain was isolated on MRS agar from the gastrointestinal (GI) of C. carpio and identified as Lysinibacillus macroides using molecular marker 16S rRNA gene. Various probiotic properties such as acid and bile tolerance and antibiotic susceptibility were analysed under in vitro conditions. Further, formulate pelletized feed using probiotic (L. macroides) in different concentrations (2, 4, 6 and 8%). Rearing of C. carpio was carried out 45 days and fed with formulated feed. The highest length (5.14 ± 0.07 cm) and weight (3.56 ± 0.07 g) of C. carpio fingerlings was recorded in the 8% LM probiotic pelletized feed, while in fingerlings fed with control showed lower in the length (3.02 ± 0.13 cm) and the weight (0.92 ± 0.04 g) on the 45th day of the experiment. Both percentage of weight gain (PWG) and specific growth rate (SGR) were significantly increased (P < 0.05) of C. carpio fingerlings fed with probiotic feed compared to control feed. Hence, the use of probiotic bacteria could be an encouraging alternative feed for future endeavours in the field of aquaculture. In conclusion, L. macroides can serve as probiotic for sustainable, competitive and promising beneficial bacteria to aquaculture industry.

Optimisation of treatment conditions for reducing Shewanella putrefaciens and Salmonella Typhimurium on grass carp treated by thermoultrasound-assisted plasma functionalized buffer.

A novel method of thermoultrasound-assisted plasma functionalized buffer (PFB) for decontaminating grass carp was evaluated using the Box-Behnken design (BBD) with processing variables including PFB generating voltage (PV), ultrasound treatment time (UT) and temperature (TP). The predicted models were found to be significant (p < 0.05) and displayed sufficient fitness with experimental data as indicated by non-significant (p > 0.05) lack of fit and high coefficient of determination (R2≥0.97) values. The optimum decontamination conditions for the responses of S. putrefaciens and S. Typhimurium were PV of 66 V, UT of 14.90 min and TP of 60 ℃, achieving reductions of 4.40 and 3.97 log CFU/g, respectively, with a desirability of 0.998. Among the variables, temperature presented higher significance for inactivating bacteria and the production of volatile basic nitrogen and lipid peroxidation under the optimized conditions were within the limits of freshness for grass carp. Additionally, the effects of PFB and the optimized thermoultrasound-assisted PFB decontamination were mild on the microstructure of grass carp with slight ruptures and loose myofibril structures, indicating the potential of thermoultrasound-assisted PFB for seafood products decontamination with reduced processing time.

The mutated Bacillus amyloliquefaciens strain shows high resistance to Aeromonas hydrophila and Aeromonas veronii in grass carp.

Bacillus amyloliquefaciens X030 (BaX030) has broad-spectrum antibacterial activity against the fish pathogens Aeromonas hydrophila and Aeromonas veronii. To improve its antibacterial effect, BaX030 was subjected to compound mutagenesis of atmospheric and room temperature plasma (ARTP) and nitrosoguanidine (NTG). The results showed that, compared with the original strain, the production of macrolactin A and oxydifficidin in mutated strain N-11 increased to 39 % and 268 %, respectively. The re-sequencing analysis suggested that there were SNPs and InDels in the gene clusters focused on the sucrose utilization pathway, glycolysis pathway and fatty acid synthesis pathway. Scanning electron microscopy revealed that strain N-11 became thin and long. The qRT-PCR results indicated that the expression of immune factors in the liver or kidney tissue of grass carp increased after feeding with N-11. H&E staining and protection experiments also showed that the mortality and surface symptoms of grass carp infected by the two pathogens were significantly reduced. The study identified a probiotic strain with potential application value in aquaculture production and provided a new strategy for the discovery of new strains with higher antibacterial biological activity.

Brachybacterium subflavum sp. nov., a novel actinobacterium isolated from the foregut of grass carp.

A novel actinobacterium, designated CFH 10395T, was isolated from the foregut of grass carp (Ctenopharyngodon idella), which had been fed with ginseng extract supplement. The taxonomic position was investigated by a polyphasic approach. Cells of CFH 10395T were Gram-staining-positive, aerobic, ovoid-shaped, non-spore-forming and non-motile. On the basis of the results of 16S rRNA gene sequence analysis, CFH 10395T was most closely related to Brachybacterium endophyticum KCTC 49087T, Brachybacterium squillarum JCM 16464T and Brachybacterium paraconglomeratum JCM 17781T (97.85%, 97.51 and 97.29% similarity, respectively). CFH 10395T grew at 4-37 °C, pH 5.0-9.0 and in the presence of up to 10.0 % NaCl (w/v). The dominant menaquinone was MK-7. The whole-cell sugars were rhamnose, glucose, mannose and galactose. meso-diaminopimelic acid was the diagnostic diamino acid in the cell-wall peptidoglycan. The major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The genome size was 3.99 Mbp with a DNA G+C content of 71.9 mol%. On the basis of the results of phylogenetic analysis, physiological properties, chemotaxonomic characteristics, low average nucleotide identity (ANI) and digital DDH (dDDH) results [ANI calculated using MUMmer (ANIm) <87 %, ANI calculated using blast (ANIb) <83 % and dDDH <23 %], it is concluded that CFH 10395T represents a novel species of the genus Brachybacterium, for which the name Brachybacterium subflavum sp. nov., is proposed. The type strain is CFH 10395T (=CGMCC 1.13804T=KCTC 49235T).