Dietary Bacillus velezensis R-71003 and sodium gluconate improve antioxidant capacity, immune response and resistance against Aeromonas hydrophila in common carp.

This study aimed to evaluate the effects of dietary supplementation with Bacillus velezensis R-71003 combined with sodium gluconate on antioxidant capacity, immune response and resistance against Aeromonas hydrophila in common carp. In addition, the biocontrol potential of the secondary metabolites of B. velezensis R-71003 was also evaluated to analyze the possible mechanism of B. velezensis R-71003 against A. hydrophila. The results indicated that the antibacterial crude extract of B. velezensis R-71003 can destroy the cell wall of A. hydrophila. Moreover, the results showed that dietary B. velezensis R-71003 could promote antioxidant capacity, which significantly increased the activities of CAT and SOD and decreased the content of MDA. Additionally, B. velezensis R-71003 supplementation significantly enhanced the immunity of common carp, as measured by the mRNA expression levels of cytokine-related genes (TNF-α, TGF-ß, IL-1ß and IL-10). In addition, dietary B. velezensis R-71003 exhibited an upregulation of IL-10 and a downregulation of IL-1ß, coupled with higher survival rates when challenged with A. hydrophila compared to the positive group. Furthermore, compared to prechallenge, the mRNA expression levels of TLR-4, MyD88, IRAK1, TRAF6, TRIF and NF-κB in the head kidney of common carp were significantly increased after challenge. The fish fed the B. velezensis R-71003 diet showed lower expression of TLR-4, MyD88, IRAK1, TRAF6, TRIF and NF-κB after the challenge than those fed the control diet. Thus, this study revealed that B. velezensis R-71003 can improve the resistance of common carp to pathogenic bacteria by destroying bacterial cell walls and improving fish immunity by activating the TLR4 signaling pathway. Importantly, this study indicated that sodium gluconate has a positive effect on B. velezensis R-71003 in enhancing the anti-infection ability of common carp. The results of this study will lay the foundation for the application of B. velezensis R-71003 in combination with sodium gluconate as an alternative to antibiotics in aquaculture.

The antipsychotic drug olanzapine altered lipid metabolism in the common carp (Cyprinus carpio L.): Insight from the gut microbiota-SCFAs-liver axis.

Olanzapine (OLA) is a common drug used to treat schizophrenia and has recently come under increasing scrutiny as an emerging contaminant. However, its impact on lipid metabolism in fish and its mechanisms of action are not well understood. In this study, common carp were exposed to 0, 10, 100, and 250 µM OLA for 60 days. The results indicated that OLA exposure increased weight gain, total cholesterol (TC), low-density lipoprotein (LDL), and triglycerides (TG) and decreased high-density lipoprotein (HDL). In addition, lipids accumulated in the liver of the common carp. To explore the underlying mechanisms of action, gut microbiota, short-chain fatty acids (SCFAs), liver transcripts, and genes related to lipid metabolism were measured. It was discovered that OLA exposure altered the common carp gut microbiota composition and increased the abundance of SCFA-producing bacteria. Correspondingly, this study showed that OLA exposure increased the levels of SCFAs, which are highly relevant to the development of lipid accumulation. Transcriptome sequencing results indicated that OLA exposure could change lipid metabolism signalling pathways, including steroid biosynthesis, the PPAR signalling pathway, asglycerophospholipid metabolism, glycerolipid metabolism, and fatty acid metabolic pathways of the common carp. Additionally, OLA exposure interrupted lipid metabolism by means of significant upregulation of lipid synthesis-related genes, including pparγ, srebp1, and fas. OLA exposure also resulted in significant lipolysis-related gene downregulation, including cpt, lpl, hsl, and pparα. The results of this study indicated that contamination of aquatic environments with OLA alters lipid metabolism in common carp. In addition, the underlying mechanism might be due in part to the modulation of the gut microbiota-SCFA-PPAR signalling pathway.

Fatty hepatocytes-derived exosomal miR-122 reduces immune function and antioxidant defence in Ctenopharyngodon idella kidney (CIK) cells.

Exosomes are important for intercellular "cross talk", but the role of exosomes in communication between hepatocytes and C. idella kidney (CIK) cells remains unknown. In this study, we detected the changes in factors related to immune and oxidative stress to investigate the molecular mechanism by which fatty hepatocyte-derived exosomes (OA-Exos) reduced immunity and induced oxidative stress in CIK cells. After incubation of CIK cells by OA-Exos for 24 h, tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB) and interleukin-1ß (IL-1ß) were significantly upregulated in the OA-Exos group (P < 0.05), and Mn superoxide dismutase (Mn-SOD) and heme oxygenase-1 (HO-1) were significantly downregulated (P < 0.05). Surprisingly, miR-122 expression was also significantly elevated after OA-Exos incubation. We further identified the expression of miR-122 and found that it was notably increased in OA-Exos compared to hepatocyte-derived exosomes (Exos). Then we transfected CIK cells with miR-122 mimic, consistently, the expression of inflammatory cytokines was also significantly elevated (P < 0.05), and the expression of glutathione peroxidase (GPx), HO-1, and Mn-SOD were dramatically decreased (P < 0.05). Furthermore, HO-1 was improved to be a direct target of miR-122, and transfection with HO-1 siRNA indicated that changes in inflammatory cytokines and genes related to oxidative stress were consistent with the above results of CIK cells incubated with OA-Exos and miR-122 mimic. We concluded that OA-Exos may, through the miR-122/HO-1 pathway, reduce immune function and antioxidant defence in CIK cells.

The Regulatory Role of Apelin on the Appetite and Growth of Common Carp (Cyprinus Carpio L.).

Apelin, a kind of active polypeptide, has many biological functions, such as promoting food intake, enhancing immunity, and regulating energy balance. In mammals, studies have indicated that apelin is involved in regulating food intake. However, there are relatively few studies about the regulatory effect of apelin on fish feeding, and the specific mechanism is not clear. Therefore, the purpose of this study was to preliminarily investigate the regulatory effects of apelin on key genes of feeding and growth in common carp (Cyprinus Carpio L.) through in vitro and in vivo experiments. In the present study, after incubation with different concentrations of Pyr-apelin-13 (0, 10, 100, and 1000 nM) in hypothalamic fragments, the expressions of Neuropeptide Y (NPY) and Agouti related peptide (AgRP) mRNA were significantly up-regulated at 12 and 3 h, respectively, and the significant down-regulation of Cocaine and amphetamine-related transcript (CART) mRNA expression was observed at 1 and 3 h. In vivo, after Pyr-apelin-13 oral administration (0, 1, 10, and 100 pmol/g), the orexin mRNA level in the hypothalamus of common carp was significantly increased at 1, 6, and 12 h, while CART/(Proopiomelanocortin) POMC mRNA levels in the hypothalamus of common carp were significantly down-regulated. Following incubation with different concentrations of Pyr-apelin-13 (0, 10, 100, and 1000 nM) in primary hepatocytes, GHR (Growth hormone receptor), IGF2 (Insulin-like growth factor 2), IGFBP2 (Insulin like growth factor binding protein 2), and IGFBP3 (Insulin like growth factor binding protein 3) mRNA levels were significantly increased at 3 h. In vivo, the levels of IGF1 (Insulin-like growth factor 1), IGF2, IGFBP2 (Insulin like growth factor binding protein 2), and IGFBP3 mRNA were significantly increased after the oral administration of Pyr-apelin-13 in the hepatopancreas, in a time and dose-dependent manner. These results support the hypothesis that Pyr-apelin-13 might regulate the feeding and growth of common carp through mediating the expressions of appetite- and growth-related genes. Overall, apelin, which is an orexigenic peptide, improves food intake and is involved in the growth of common carp.

Impact of chronic exposure to the ionic liquid ([C8mim][PF6]) on intestinal physical barrier, immunological barrier and gut microbiota in common carp (Cyprinus carpio L.).

Ionic liquids (ILs) are commonly known as "green" solvents and have been widely used in various fields. However, the ecotoxicity of ILs in aquatic environment has received considerable attention from scientific researchers. This study investigated the toxic effects of different concentrations of 1-octyl-3-methylimidazolium hexafluorophosphate ([C8mim][PF6]) (0, 1.35, 2.70 and 5.40 mg/L) on intestinal physical barrier, immunological barrier, and intestinal microbiome in common carp on days 30 and 60. The results showed that ([C8mim][PF6]) exposure could reduce the intestinal villus height, decrease the mRNA expression of tight junction genes (occludin, claudin-2 and zonula occludens-1), and increase the levels of D-lactic and diamine oxidase, and reduce acid phosphatase and lysozyme activities, complement 3 and 4 contents, and anti-inflammatory cytokine TGF-ß protein level, while increase pro-inflammatory cytokines TNF-α and IL-1ß protein levels in common carp. Moreover, ([C8mim][PF6]) exposure was also found to significantly reduce gut microbial diversity and alter microbial community structure in common carp. Collectively, our study highlighted that exposure to ([C8mim][PF6]) could disrupt intestinal physical barrier, impair immunological barrier and alter intestinal microbiome in common carp, suggesting that ILs exert a negative effect on fish intestinal health status and may pose serious health risks in fish. The results of this study may be helpful to illuminate the toxicity mechanisms of the ILs on fish.

Phylogeny of Slc15 family and response to Aeromonas hydrophila infection following Lactococcus lactis dietary supplementation in Cyprinus carpio.

Solute carrier 15 family (Slc15) are membrane proteins that utilize the proton gradient and negative membrane protential for the transmembrane transporter of di-/tripeptide and peptide-mimetic molecules, in addition, they also play important roles in immunoreaction. In this study, 10 Slc15 genes were identified in the common carp genome database. Comparative genomics analysis showed considerable expansion of the Slc15 genes and verified the four-round whole genome duplication (WGD) event in common carp. Phylogenetic analysis revealed all Slc15 genes of common carp were clustered into orthologous groups indicating the highly conservative during evolution. Besides, the tissues and temporal expression examined by RT-PCR and qRT-PCR showed that most of the Slc15 genes had a narrow tissue distribution and exhibited tissue-specific expression patterns. Expression divergences were observed between these copies proving function divergence after the WGD. Then, we investigated the dietary supplementation effects of three Lactococcus lactis strains on the expression of Slc15 genes in common carp infected by A. hydrophila to find an effective way to treat aquatic diseases. Almost all of the Slc15 genes had an increased expression trend in the early post-challenge stage, and reached the highest expression level at 12h post-challenge. Then, the expression level showed a bluff descent at the last two stages and the expression level reached the lowest at 48 h post-challenge. Slc15 genes expression is actively up-regulated when stimulated by inflammatory factors, which can "amplify" immune signals, and improve the body's defense against foreign invasion in the early stage of the inflammatory response. So activation of the Slc15 genes may be an effective way for infectious disease treatment. As expected, three strains improved the expression of Slc15 genes variously compared with the control/infection groups. The strain 3 of L. lactis had a better induction of Slc15 genes compared with strain 1 and strain 2. It might be applied as a potential activation of Slc15 genes for disease treatment and adding befitting L. lactis may be a good way to protect aquatilia from bacillosis.

The Effects of Oral Rehmannia glutinosa Polysaccharide Administration on Immune Responses, Antioxidant Activity and Resistance Against Aeromonas hydrophila in the Common Carp, Cyprinus carpio L.

The effects of the oral administration of Rehmannia glutinosa polysaccharide (RGP-1) on the immunoregulatory properties, antioxidant activity, and resistance against Aeromonas hydrophila in Cyprinus carpio L. were investigated. The purified RGP-1 (250, 500, and 1,000 µg/mL) was co-cultured with the head kidney cells of the common carp. The proliferation and phagocytosis activities of the head kidney cells, and the concentration of nitric oxide (NO) and cytokines in the culture medium were determined. Next, 300 common carps (47.66 ± 0.43 g) were randomly divided into five groups; the two control groups (negative and positive) were administered sterile PBS and the three treatment groups were administered different concentrations of RGP-1 (250, 500, and 1,000 µg/mL) for seven days. Subsequently, the positive and treatment groups were infected with A. hydrophila, and the negative group was administered sterile PBS for 24 h. The concentration of NO, cytokines, lysozyme (LZM), and alkaline phosphatase (AKP) in serum, the total antioxidant capacity (T-AOC), the levels of malonaldehyde (MDA) and glutathione (GSH), and the total activities of superoxide dismutase (T-SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in the hepatopancreas of the common carp were tested. We observed that RGP-1 could significantly enhance the proliferation and phagocytosis activities (P < 0.05), besides inducing the production of NO, pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, IL-12) and anti-inflammatory cytokines (IL-10, TGF-ß) (P < 0.05) in vitro. The in vivo experimental results revealed that RGP-1 significantly enhanced NO production, protein levels of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, IL-12), LZM and AKP activities, and the antioxidant content (T-AOC, SOD, CAT, GSH, GSH-Px, and MDA) compared to that observed in the negative group prior to A. hydrophila infection (P < 0.05). NO, pro-inflammatory cytokines, LZM and AKP activities were significantly lower than that in the positive group after infection (P < 0.05). However, whether infected or not, the expression of anti-inflammatory cytokines (IL-10, TGF-ß) increased significantly in the RGP-1-treated groups (P < 0.05). Therefore, the results suggested that RGP-1 could enhance the non-specific immunity, antioxidant activity and anti-A. hydrophila activity of the common carp, and could be used as a safe and effective feed additive in aquaculture.

Effects of an exopolysaccharide from Lactococcus lactis Z-2 on innate immune response, antioxidant activity, and disease resistance against Aeromonas hydrophila in Cyprinus carpio L.

Microbial exopolysaccharides (EPS) from Lactococcus have been found to have an important role in the probiotic activity of this bacterium; however, the immunomodulatory and antioxidant activities have not been fully explored in aquaculture. In the present study, we investigated EPS-2 from Lactococcus lactis Z-2, isolated from healthy common carp, for its immunomodulatory and antioxidant effects and disease resistance against Aeromonas hydrophila in Cyprinus carpio L. We found that the molecular weight of EPS-2 was 18.65 KDa. The monosaccharide composition of this polymer was rhamnose, xylose, mannose, glucose, and galactose at a molar percentage of 13.3%, 14.1%, 18.5%, 27.4%, and 26.7%, respectively. EPS-2 treatment could modulate the immune responses in vitro and in vivo. In vitro tests showed that EPS-2 could significantly enhance the proliferation and phagocytosis activities (P < 0.05) as well as induce the production of nitic oxide (NO), pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6), and anti-inflammatory cytokines (IL-10, TGF-ß) (P < 0.05) in head kidney cells. When the fish were gavaged with three different concentrations of EPS-2 (250, 500, 1000 µg/mL) for 7 days and infected with A. hydrophila, different expression patterns of the NO, cytokines, lysozyme (LZM), and alkaline phosphatase (AKP) in the serum and of antioxidants (T-AOC, SOD, CAT, GSH, GSH-Px and MDA) in hepatopancreas were observed. Before infection with A. hydrophila, EPS-2 supplementation significantly up-regulated the NO production, protein levels of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6), LZM and AKP activities, and levels of antioxidant molecules compared to those in the negative (G1) group (P < 0.05), whereas levels of NO and pro-inflammatory cytokines and LZM and AKP activities were significantly lower than those in the positive (G2) group after infection (P < 0.05). However, whether infected or not, the expression levels of anti-inflammatory cytokines (IL-10, TGF-ß) were significantly increased in the EPS-2 treatment groups (P < 0.05). These results indicate that EPS-2 has immunomodulatory and antioxidant effects on common carp, both in vitro and/or in vivo, and can be applied as a common carp feed supplement to enhance fish immunity and disease resistance against A. hydrophila.

Impact of chronic exposure to trichlorfon on intestinal barrier, oxidative stress, inflammatory response and intestinal microbiome in common carp (Cyprinus carpio L.).

Trichlorfon is an organic phosphorus pesticide used to control different parasitic infections in aquaculture. The repeated, excessive use of trichlorfon can result in environmental pollution, thus affecting human health. This study aimed to determine the effects of different concentrations of trichlorfon (0, 0.1, 0.5 and 1.0 mg/L) on the intestinal barrier, oxidative stress, inflammatory response and intestinal microbiome of common carp. Trichlorfon exposure significantly reduced the height of intestinal villus and decreased the expression levels of tight junction genes, such as claudin-2, occludin and ZO-1, in common carp. Moreover, the activities of antioxidant enzymes, such as CAT, SOD and GSH-Px, exhibited a decreasing trend with increasing trichlorfon concentrations, while the contents of MDA and ROS elevated in the intestinal tissues of common carp. The mRNA and protein levels of pro-inflammatory cytokines TNF-α and IL-1ß were significantly upregulated by trichlorfon exposure. The level of anti-inflammatory cytokine TGF-ß was remarkably higher in 1.0 mg/L trichlorfon treatment group compared to control group. In addition, the results demonstrated that trichlorfon exposure could affect the microbiota community composition and decreased the community diversity in the gut of common carp. Notably, the proportions of some probiotic bacteria, namely, Lactobacillus, Bifidobacterium and Akkermansia, were observed to be reduced after trichlorfon exposure. In summary, the findings of this study indicate that exposure to different concentrations of trichlorfon can damage intestinal barrier, induce intestinal oxidative damage, trigger inflammatory reaction and alter gut microbiota structure in common carp.

Characterization of a polysaccharide HP-02 from Honeysuckle flowers and its immunoregulatory and anti-Aeromonas hydrophila effects in Cyprinus carpio L.

Aquaculture systems are known to be major reservoirs of Aeromonas hydrophila. The use of Chinese medicinal polysaccharides has become a successful strategy to prevent bacterial diseases in aquaculture. A water-soluble polysaccharide, HP-02, was purified from honeysuckle flowers, and the present study was aimed to examine its immunomodulatory and anti-A. hydrophila effects in Cyprinus carpio L. The results showed that the molecular weight of HP-02 was estimated to be 38 kDa. Monosaccharide composition was determined to be arabinose, rhamnose, mannose, glucose, and galactose in a molar ratio of 2.5: 1.8: 3.6: 3.7: 1.9. HP-02 not only distinctly improved the proliferation and phagocytosis of head kidney cells, but also exerted significant immunoregulation activity by increasing the secretion of the pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-12 and the anti-inflammatory cytokines IL-10 and TGF-ß at the protein level in head kidney cells and serum. Moreover, during pathogen infection in vivo, HP-02 significantly reduced the expression of pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokines. These results indicated that HP-02 had immunomodulatory effects on common carp both in vitro and in vivo, and HP-02 can be applied as a common carp feed supplement to enhance fish immunity and disease resistance against A. hydrophila.

Effects of Lactococcus lactis from Cyprinus carpio L. as probiotics on growth performance, innate immune response and disease resistance against Aeromonas hydrophila.

In the present study, we reported 18 LAB strains isolated from the intestinal contents of Cyprinus carpio, and their probiotic properties both in vitro and in vivo. The results showed that 9 of them had higher in vitro immunomodulatory properties, effectively survived under acidic (pH 2.5) and bile salt (ranging from 0.1% to 0.5%) conditions, and inhibited the growth of 4 pathogens. Among them, Lactococcus lactis Q-8, Lactococcus lactis Q-9, and Lactococcus lactis Z-2 showed the strongest adhesion abilities and inhibition of pathogen adhesion to mucin. When the fish consumed diets containing these 3 strains (5 × 108 CFU/g) for 8 weeks, the weight gain (WG) and specific growth rate (SGR) had significantly (P < 0.05) increased, especially with L. lactis Q-8, which had a WG of 231.45%, and SGR of 2.22%. Survival rate in each LAB supplementation group was also significantly higher than that in control group during the feeding period (P < 0.05). For the cytokines expression levels in serum, different expression patterns were also observed. Before the infection with Aeromonas hydrophila, L. lactis supplementation significant up-regulated protein levels of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, IL-12) compared with negative (CK1) group, while these cytokines were significantly lower than those in positive (CK2) group after infection. However, whether infected or not, the expression of anti-inflammatory cytokines (IL-10, TGF-ß) were significantly increased in L. lactis Q-8, L. lactis Q-9, and L. lactis Z-2 treatment groups. In conclusion, these 3 L. lactis strains screened from common carp were effective in improving growth, innate immunity and disease resistance. Based on the physiological characteristics in our study, they might be used as potential probiotics in aquaculture.

Effects of dietary supplementation of three strains of Lactococcus lactis on HIFs genes family expression of the common carp following Aeromonas hydrophila infection.

HIFs (Hypoxia inducible factors) are the main regulators of the expression change of oxygen-dependent genes, in addition, they also play important roles in immune regulation. HIFs participate in infectious diseases and inflammatory responses, providing us a new therapeutic target for the treatment of diseases. In this study, 16 HIFs were identified in common carp genome database. Comparative genomics analysis showed large expansion of HIF gene family and approved the four round whole genome duplication (WGD) event in common carp. To further understand the function of HIFs, the domain architectures were predicted. All HIF proteins had the conserved HLH-PAS domain, which were essential for them to form dimer and bind to the downstream targets. The differences in domain of HIFα and HIFß might result in their different functions. Phylogenetic analysis revealed that all HIFs were divided into two subfamilies and the HIFs in common carp were clustered with their teleost counterparts indicating they are highly conservative during evolution. In addition, the tissue distribution was examined by RT-PCR showed that most of HIF genes had a wide range of tissue distribution but exhibited tissue-specific expression patterns. The expression divergences were observed between the copy genes, for example, HIF1A-1, HIF2A-1, ARNT-2 had wide tissue distribution while their copies had limited tissue distribution, proving the function divergence of copies post the WGD event. In order to find an effective activation of HIFs and apply to treatment of aquatic diseases, we investigate the dietary supplementation effects of different strains of Lactococcus lactis on the expression of HIFα subfamily members in kidney of common carp infected with A. hydrophila. In addition, all of the HIF genes have a high expression in the early stages of infection, and decreased in the treatment time point of 48 h in common carp. This phenomenon confirms that as a switch, the main function of HIFs is to regulate the production of immune response factors in early infection. So activation of the switch may be an effective method for infectious disease treatment. As expected, the treatment groups improved the expression of HIFs compared with the control group, and the effects of the three strains are different. The strain1 of L. lactis had a stronger induction on HIF genes than strain2 and strain3, and it might be applied as a potential activation of HIF genes for disease treatment. So, adding befitting L. lactis maybe a well method to activate the HIF genes to protect them from mycobacterial infection.

Effects of dietary Radix Rehmanniae Preparata polysaccharides on the growth performance, immune response and disease resistance of Luciobarbus capito.

This work explores the effects of dietary Radix Rehmanniae Preparata polysaccharide (RRPP) supplementation on the growth performance, nonspecific immune responses, immune- and growth-related gene expression and disease resistance to Aeromonas hydrophila in Luciobarbus capito. Diets containing five concentrations of 0%, 0.05%, 0.1%, 0.2% and 0.4% RRPP were fed to fish for 60 d. The results indicated that the growth performance significantly increased in the 0.1%, 0.2% and 0.4% RRPP groups compared with that in the control (P < 0.05). The activities of serum lysozyme (LAZ), acid phosphatase (ACP), superoxide dismutase (SOD), alkaline phosphatase (AKP) and total protein (TP) were significantly increased in the appropriate RRPP supplemented groups (P < 0.05). With respect to immune- and growth-related genes, such as interleukin (IL)-1ß, IL-8, tumor-necrosis factor (TNF)-α, interferon (IFN)-γ, growth hormone (GH), insulin-like growth factor (IGF)-I and IGF-II, up-regulation were observed in the three organs (kidney, spleen, gut) of the fish fed with RRPP, compared with the control. In contrast, the mRNA expression of IL-10 and transforming-growth factor (TGF)-ß were downregulated. After challenge with A. hydrophila, the final survival rate was significantly higher in fish fed the RRPP supplement than that in the control group (P < 0.05). In conclusion, RRPP enhanced the growth performance, immune response and disease resistance of Luciobarbus capito, with the greatest effects at 0.2% RRPP.

Effects of cadmium exposure on the composition and diversity of the intestinal microbial community of common carp (Cyprinus carpio L.).

Cadmium (Cd) is an environmental pollutant that poses serious health hazards. Due to the increasing contamination of aquatic systems with Cd, the increased accumulation of Cd in fish has become a food safety and public health concern. The present study was conducted to investigate the effects of waterborne Cd exposure on the microbial community composition and diversity in the gut of common carp. Common carp were exposed to three waterborne Cd concentrations (0, 50 and 500 µg Cd L-1) for 4 weeks. Our results indicated that Cd exposure profoundly affected the composition of the gut microbiota in the common carp. At the phylum level, Saccharibacteria were detected in only the 0 µg and 50 µg Cd L-1 exposure groups, and the abundance of Fusobacteria decreased with increasing Cd concentration, while the abundance of Firmicutes increased with increasing Cd concentration. At the genus level, Cetobacterium was the dominant group in the gut of the common carp, and the abundance of Cetobacterium decreased after Cd exposure. Notably, the abundance of Akkermansia muciniphila, a probiotic, was found to decrease after Cd exposure, and the proportions of some Cd-resistant bacteria were found to increase following Cd exposure. Our results also demonstrated that Cd exposure decreased the community diversity of the gut microbiota. These results suggest that Cd exposure may impact the gut homeostasis of common carp and further affect the health of the organism.

Dietary supplementation with Rehmannia glutinosa affects the composition of intestinal microorganisms in common carp.

A feeding experiment was conducted to investigate the effects of Rehmannia glutinosa (RG) on the bacterial, archaeal, and fungal community composition in the gut of common carp (Cyprinus carpio L.). Common carp were given a control diet and an RG-supplemented diet (basal diet plus 4% prepared Rehmannia root powder) over a period of 80 days. Our results indicated that the bacteria Fusobacteria, Proteobacteria, and Bacteroidetes; the archaea Crenarchaeota and Euryarchaeota; and the fungi Basidiomycota are the most abundant microbial taxa in the gut of common carp. Compared with the common carp fed a control diet, the common carp fed an RG-supplemented diet contained a higher content of Akkermansia sp., and a lower proportion of Aeromonas sp. These results indicate that the consumption of a diet containing RG can lead to the accumulation of more beneficial microorganisms while inhibiting the growth of potential pathogens. Moreover, Crenarchaeota, the methanogenic, and Basidiomycota were detected in this study, these populations may be of high physiological relevance in carp because they have been implicated in human health and disease. Our results suggest that an RG-supplemented diet changes the intestinal microbial composition of common carp, which could have positive effects on the immune response of carp.

Lactobacillus plantarum Restores Intestinal Permeability Disrupted by Salmonella Infection in Newly-hatched Chicks.

Salmonella infections in newly hatched chicks result in enteric and systemic diseases with a high mortality. Probiotics can improve the health of a host. The purpose of the present study was to investigate the effect of Lactobacillus plantarum LTC-113 on the gut permeability in the presence or absence of Salmonella (Salmonella Typhimurium) infection. Newly hatched chicks were randomly allocated to 4 treatments (i) NC (negative control); (ii) LAC (the L. plantarum LTC-113-treated group); (iii) SAL (the Salmonella-infected group), and (iv) LAC + SAL (the L. plantarum LTC-113-treated and Salmonella-infected group). Compared with the NC group, the intestinal permeability and claudin-2 (CLDN-2) were significantly increased, while mRNA levels of zonula occludens-1 (ZO-1) and claudin-5 (CLDN-5) were significantly decreased in the SAL group. However, these changes were eliminated in the LAC + SAL group. Additionally, numbers of Salmonella in liver, spleen and ceca were significantly reduced in the LAC + SAL group compared with the SAL group. Moreover, L. plantarum LTC-113 prevented the increase of inflammatory meditators myeloperoxidase (MPO), LITAF, IL-1ß, IL-6 and inflammation scores induced by Salmonella. These findings indicate that L. plantarum LTC-113 can protect hosts from Salmonella induced intestinal barrier disruption by regulating expression of tight junction genes and inflammatory meditators and decreasing Salmonella colonization.

Molecular characterization and dietary regulation of aminopeptidase N (APN) in the grass carp (Ctenopharyngodon idella).

Aminopeptidase N (APN) is a member of the peptidase M1 family and plays an important role in protein digestion. In the present study, an APN gene was cloned from the intestine of Ctenopharyngodon idellus. The full-length cDNA sequence of APN encodes an 892-amino-acid peptide that includes one helix trans-membrane region. Phylogenetic analysis showed that the APN sequence clustered with Danio rerio as its closest neighbor, sharing a sequence similarity of 81.5%. APN mRNA was differentially expressed in different tissues, with a gradient expression from high to low in the tissues of the fore-intestine, hind-intestine, liver, mid-intestine, kidney, muscle, spleen and heart. APN expression in grass carp had a circadian pattern, showing time-dependent higher expression between 06:00 and 18:00 and lower expression between 18:00 and 06:00. In addition, the protein levels and resource in the diet-regulated APN expression suggested that low crude protein (CP) level and fish meal stimulated APN gene expression. Furthermore, the mRNA expression of APN in the intestine was significantly suppressed by high concentrations of glutamine and glutamine dipeptides, respectively. This study may provide valuable knowledge on the regulation of APN expression in teleost, which has potential applications for improving fish dietary formulations.

Using In Vitro Immunomodulatory Properties of Lactic Acid Bacteria for Selection of Probiotics against Salmonella Infection in Broiler Chicks.

Poultry is known to be a major reservoir of Salmonella. The use of lactic acid bacteria has become one of successful strategies to control Salmonella in poultry. The purpose of this study was to select lactic acid bacteria strains by their in vitro immunomodulatory properties for potential use as probiotics against Salmonella infection in broiler chicks. Among 101 isolated lactic acid bacteria strains, 13 strains effectively survived under acidic (pH 2.5) and bile salt (ranging from 0.1% to 1.0%) conditions, effectively inhibited growth of 6 pathogens, and adhered to Caco-2 cells. However, their in vitro immunomodulatory activities differed significantly. Finally, three strains with higher in vitro immunomodulatory properties (Lactobacillus plantarum PZ01, Lactobacillus salivarius JM32 and Pediococcus acidilactici JH231) and three strains with lower in vitro immunomodulatory activities (Enterococcus faecium JS11, Lactobacillus salivarius JK22 and Lactobacillus salivarius JM2A1) were compared for their inhibitory effects on Salmonella adhesion and invasion to Caco-2 cells in vitro and their antimicrobial effects in vivo. The former three strains inhibited Salmonella adhesion and invasion to Caco-2 cells in vitro, reduced the number of Salmonella in intestinal content, spleen and liver, reduced the levels of lipopolysaccharide-induced TNF-α factor (LITAF), IL-1ß, IL-6 and IL-12 in serum and increased the level of IL-10 in serum during a challenge study in vivo more efficiently than the latter three strains. These results suggest that in vitro immunomodulatory activities could be used as additional parameters to select more effective probiotics as feed supplements for poultry.

Screening of immunomodulatory and adhesive Lactobacillus with antagonistic activities against Salmonella from fermented vegetables.

The purpose of this study was to select strains of lactic acid bacteria (LAB) by their in vitro adhesive and immunomodulatory properties for potential use as probiotics. In this study, 16 randomly selected LAB strains from fermented vegetables (sauerkraut, bean and cabbage) were first screened for their tolerance to acid, bile salts, pepsin and pancreatin, bacterial inhibitory activities and abilities to adherence to Caco-2 cells. Then, 4 strains with the highest adhesion abilities were selected for further studies of their immunomodulatory properties and inhibitory effects against Salmonella adhesion and invasion to Caco-2 cells in vitro. The results showed that these 16 LAB strains effectively survived in simulated gastrointestinal condition and inhibited growth of six tested pathogens. Lactobacillus rhamnosus P1, Lactobacillus plantarum P2, Lactobacillus rhamnosus P3 and Lactobacillus casei P4 had the highest abilities to adhere to Caco-2 cells. Furthermore, L. plantarum P2 strain showed higher abilities to induce expression of tumor necrosis factor-α and interleukin-12 by splenic monocytes and strongly inhibited the adhesion and invasion of S. enteritidis ATCC13076 to Caco-2 cells. These results suggest that Lactobacillus strains P2 could be used as a probiotic candidate in food against Salmonella infection.

Characterization and dietary regulation of oligopeptide transporter (PepT1) in different ploidy fishes.

The oligopeptide transporter (PepT1) is located on the brush-border membrane of the intestinal epithelium which has been regarded as a mediator of protein absorption. Here, we cloned and characterized PepT1 genes from diploid (red crucian carp), triploid and tetraploid fish. Then, the PepT1 expression pattern in different tissues and embryogenesis were assayed. Meanwhile, using real-time PCR and western blotting, we showed the expression profiles of diets with different protein levels, protein sources and additives (sodium butyrate) in triploids. The cDNAs of the three different ploidy fishes have a high sequence similarity of PepT1 among vertebrates. PepT1 mRNA expression was also developmentally regulated and showed the strongest expression around the 2-cell and 4-cell stage in all three kinds of fishes. The maternal transcripts were first detected in eggs and dropped from blastula stage to muscle contraction stage. Tissue expression studies showed higher expression of PepT1 genes in the intestines of fishes compared with other tissues. In adults, triploids showed significantly higher expression levels of PepT1 in the intestines of the three kinds of ploidy fishes during breeding season and non-breeding season. In addition, high or low protein level diets both promote PepT1 expression in the intestine. We also confirmed that fish meal showed a significant increase in PepT1 expression than soybean meal in triploid intestines. Furthermore, sodium butyrate additives induce PepT1 expression that may be mediated by CDX2 and CREB. This research provides a new insight into protein absorption and its regulation in triploid fish.