Effect of Bacillus amyloliquefaciens LSG2-8 on the intestinal barrier function of Amur minnow (Rhynchocypris lagowskii).

Probiotics are widely used in aquaculture. This article aims to study the effect of Bacillus amyloliquefaciens LSG2-8 on the intestinal barrier function of Rhynchocypris lagowskii. B. amyloliquefaciens LSG2-8 were added to R. lagowskii basal diets (CK) as additives at four concentrations: 1.0 × 106 (D-6), 1.0 × 107 (D-7), 1.0 × 108 (D-8) and 1.0 × 109 (D-9) CFU g-1 by dry weight of basal diet. After a 56-day feeding experiment, the activities of intestinal digestive enzymes and immunity-related enzymes of R. lagowskii on group D-6, D-7, D-8 and D-9 diet were significantly higher than the control (P < 0.05). In molecular experiments, the authors found that the levels of TGF-ß mRNA, IL-10 mRNA, ZO-1 mRNA and claudin-3 mRNA in group D-8 R. lagowskii were significantly higher (P < 0.05) than those of the control and other groups. Furthermore, the levels of IL-1ß and IL-8 mRNA of R. lagowskii on group D-6, D-7, D-8 and D-9 diet were significantly lower than those of the control (P < 0.05). In addition, the authors found that B. amyloliquefaciens LSG2-8 can regulate the intestinal flora balance and improve the intestinal structure of R. lagowskii. In conclusion, B. amyloliquefaciens LSG2-8 can improve the intestinal barrier function of R. lagowskii and can be used as a feed additive in aquaculture.

The immuneoreaction and antioxidant status of Chinese mitten crab (Eriocheir sinensis) involve protein metabolism and the response of mTOR signaling pathway to dietary methionine levels.

To study the effects of dietary methionine on growth performance, immunity, antioxidant capacity, protein metabolism, inflammatory response and apoptosis factors in Chinese mitten crabs (Eriocheir sinensis). Five diets with different methionine levels (0.63%, 0.85%, 1.06%, 1.25% and 1.47%) were fed to E. sinensis for 8 weeks. Results showed that in the 1.25% Met group, both growth performance and feed utilization were significantly increased. The crude protein content of crab muscle in the 1.06% and 1.25% Met groups was significantly higher than that in the control group. The immune and antioxidant enzyme activities, as well as gene expression levels of anti-lipopolysaccharide factor 1 (ALF1), Crustin-1, prophenoloxidase (proPO), cap 'n' collar isoform C (CncC) in 1.25% Met group were significantly higher than other groups. The activities of adenosine deaminase (ADA) and glutamate transaminase (GPT) in serum decreased first and then increased with the increase of methionine content, while the changes of ADA and GPT in hepatopancreas increased first and then decreased. 1.25% Met group exhibited significantly increased levels of GOT, GPT, and ADA compared to the control group. 1.25% Met diet group significantly up-regulated protein synthesis and anti-apoptotic factors, and significantly down-regulated inflammatory and pro-apoptotic factors in hepatopancreas. At 1.25% in the diet, methionine was found to boost E. sinensis growth, muscle protein deposition and immunity, as well as its antioxidant capacity. Combined with the above results, based on the expression of factors involved in the mammalian target of rapamycin (mTOR) signaling pathway and the mitogen-activated protein kinase (MAPK) signaling pathway, it is proved that methionine can not only promote protein metabolism, improve feed utilization, but also alleviate the inflammatory response and apoptosis caused by oxidative stress in the body.

Dietary supplementation of two indigenous Bacillus spp on the intestinal morphology, intestinal immune barrier and intestinal microbial diversity of Rhynchocypris lagowskii.

This study evaluated the effects of dietary administration of two indigenous Bacillus (A: basal control diet; B: 0.15 g/kg of Bacillus subtilis; C: 0.1 g/kg of Bacillus subtilis and 0.05 g/kg of Bacillus licheniformis; D: 0.05 g/kg of Bacillus subtilis and 0.1 g/kg of Bacillus licheniformis; E: 0.15 g/kg of Bacillus licheniformis) on the digestive enzyme activities, intestinal morphology, intestinal immune and barrier-related genes relative expression levels, and intestinal flora of Rhynchocypris lagowskii. The results showed that the fold height, lamina propria width, and muscle layer thickness of midgut and hindgut in group C were significantly higher than that of group A (P < 0.05). The activities of protease, amylase, and lipase in group C were significantly higher than those of group A (P < 0.05). The relative expression levels of IL-1ß and IL-8 in the intestine of group C were significantly downregulated, and the relative expression levels of IL-10 and TGF-ß were significantly upregulated (P < 0.05). The relative expression levels of Claudin-2 in group A significantly increased and the relative expression levels of Claudin-4 in group A significantly reduced compared with other groups (P < 0.05). The relative expression levels of ZO-1 in groups C and D were significantly higher than those of other groups (P < 0.05). The Bacillus in the intestine of group C has the highest relative abundance among all groups. Overall, it can generally be concluded that dietary supplementation of indigenous Bacillus subtilis and Bacillus licheniformis (group C) can improve the intestinal morphology, digestion, and absorption enzyme activities, enhance intestinal mucosal immunity and barrier function, and maintain the intestinal microbial balance of R. lagowskii.

Construction and immune efficacy of recombinant Lactobacillus casei strains expressing Malt from Aeromonas veronii.

Aeromonas veronii is an important zoonotic pathogen that causes significant economic losses in the aquaculture industry. The use of probiotics in aquaculture is a practical alternative to antibiotics to promote animal health and aid in disease prevention. In the present study, we aimed to construct a recombinant Lactobacillus casei(surface-displayed or secretory) strain containing Malt from A. veronii TH0426 and assess its potential as an oral vaccine. A 1314-bp Malt gene fragment was successfully amplified and cloned into a prokaryotic protein expression system. Protein expression in resulting recombinant strains Lc-MCS-Malt (surface-displayed) and Lc-pPG-Malt (secretory) was then verified by Western blotting and indirect immunofluorescence. A single band was observed on the Western blots, with the molecular weight of the corresponding protein shown to be 48 kDa. Furthermore, a fluorescent signal for Lc-MCS-Malt was observed by fluorescence microscopy. At 0, 7, 16, 25, and 34 days post-immunization, tissue and blood samples were collected from common carp orally administered with the recombinant L. casei strains for immune-related index analyses. Treatment of common carp with the recombinant vaccine candidate stimulated high serum or skin mucus specific antibody titers and induced a higher lysozyme, ACP, SOD activity, while fish fed with Lc-pPG or PBS had no detectable immobilizing immune responses. Expression of IL-10, IL-1ß, TNF-α, and IFN-γ genes in the group immunized with recombinant L. casei were significantly (P < 0.05) up regulated as compared with control groups, indicating that inflammatory response and cell immune response were triggered. Results also showed that recombinant L. casei could stimulate the mucosa through colonization of the intestine, resulting in increased transcription of IL-10, IL-1ß, TNF-α, and IFN-γ. Immunity and colonization assays also showed that after 34 days of fasting, recombinant L. casei were still present in the intestines of the immunized fish. Common carp that received Lc-MCS-Malt(53.3%) and Lc-pPG-Malt (46.7%) exhibited higher survival rates than the controls after challenge with the pathogen A. veronii. Our findings suggested that recombinant L. casei can adequately protect fish and improve immunity, providing a theoretical basis for the future development of an oral Lactobacillus vaccine for use in aquaculture.

Amelioration of hexavalent chromium-induced bioaccumulation, oxidative stress, tight junction proteins and immune-related signaling factors by Allium mongolicum Regel flavonoids in Ctenopharyngodon idella.

Chromium (Cr) is the most common heavy metal and that becomes toxic when present at higher concentrations in aquatic environments. Allium mongolicum Regel flavonoids (AMRF) has been documented to possess detoxification, antioxidant and anti-inflammatory properties. The aim of this study was to explore the potential of dietary AMRF and Cr exposure on bioaccumulation, oxidative stress, and immune response in Ctenopharyngodon idella. After acclimation, 360 fish were randomly distributed into six groups. The fish were fed with diets supplemented with Cr and/or AMRF for 4 weeks (28 days), the Cr concentrations were 0, 120, and 240 mg/kg and the concentrations of AMRF were 0 or 40 mg/kg, respectively. The results shown that Cr accumulation in the kidney, liver, spleen, intestine and gill were significantly increased following Cr exposure, dietary AMRF supplementation attenuated the increased in Cr accumulation. Dietary AMRF supplementation significantly reduced the levels of malondialdehyde (MDA) and protein carbonyl (PC) in liver, spleen and gill compared with the same Cr dose groups. When fish were supplemented with AMRF significantly increased lysozyme activity (LZM), complement 3 (C3) in kidney and intestine compared with the same Cr dose groups. Serum glutamic oxalate transaminase (GOT) and glutamic pyruvate transaminase (GPT) were significantly increased following exposure to Cr. Dietary AMRF supplementation significantly decreased GOT and GPT activity in the serum. In addition, AMRF supplementation can decrease the expression of inflammatory (NF-κB p65, TNF-α and IL-1ß) and increased the expression of tight junction proteins (occludin and ZO-1) following Cr exposure in C. idella. These results indicate that AMRF has the potential to alleviate the effects of Cr toxicity in C. idella.

Astaxanthin protects lipopolysaccharide-induced inflammatory response in Channa argus through inhibiting NF-κB and MAPKs signaling pathways.

The present study was conducted to evaluate the protective effects of astaxanthin against lipopolysaccharide (LPS)-induced inflammatory responses in Channa argus in vivo and ex vivo. Primary hepatocytes were exposed to different concentrations of LPS for 24 h to induce an inflammatory response, and the protective effects of astaxanthin against LPS-induced inflammation were studied ex vivo and in vivo. Hepatocytes exposed to LPS (5-20 µg mL-1) alone for 24 h resulted in a significant increase in lactate dehydrogenase release (LDH), Nitric oxide (NO) production and Malondialdehyde (MDA) content, 10 µg mL-1 LPS could induced inflammatory response in hepatocytes. Gene expression of TLR4, NFkBp65, MAPKp38, TNF-α, IL-6 and IL-1ß mRNA expression were also enhanced ex vivo (p < 0.05). In vivo test demonstrated that pretreatment with astaxanthin prevented the LPS-induced upregulation of pro-inflammatory cytokines TNF-α, IL-6 and IL-1ß. Besides, astaxanthin blocked the expression of Toll-like receptor 4 (TLR4) and then suppressed the phosphorylation of nuclear transcription factor-kappa B (NF-κB) p65 and degradation inhibitor of NF-κBα (IκBα). Further study showed that astaxanthin could suppress the phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) in mitogen-activated protein kinase (MAPK) signal pathway. In conclusion, our results suggest that astaxanthin played an anti-inflammatory role by regulating TLR4 and the NF-κB and MAPK signaling pathways in C. argus.

L-carnitine regulated Nrf2/Keap1 activation in vitro and in vivo and protected oxidized fish oil-induced inflammation response by inhibiting the NF-κB signaling pathway in Rhynchocypris lagowski Dybowski.

Nrf2/Keap1 pathway is associated with oxidative stress. l-carnitine is currently under preclinical evaluation as a antioxidant, but the use of l-carnitine in aquaculture has been poorly evaluated and so far no mechanism has been demonstrated. Here, we explored the effects of l-carnitine in vitro and in vivo and discussed the possible molecular mechanisms involved. Firstly, Nrf2-siRNA significantly knocked down the mRNA level of Nrf2 in FHM cells. Thus, the activities of antioxidant enzymes (T-SOD, CAT, GSH-PX) and the level of antioxidant substance (GSH) and the level of MDA showed that Nrf2-siRNA pretreatment weakened the protective effect of l-carnitine. Moreover, the mRNA levels of Keap1, Nrf2, Maf and HO-1 indicated that l-carnitine regulated Nrf2/Keap1 activation. Furthermore, oxidized fish oil remarkably suppressed growth in Rhynchocypris lagowski Dybowski, and the lower antioxidant capacity was also observed in liver. According to the results of immune related indexes (the levels of IL-1ß, TNF-α, LZM, AKP) in serum and the mRNA levels of immune related genes (NF-κB, IL-1ß, TNF-α, IL-8, IL-10 and TGF-ß) in liver, oxidized fish oil also induced inflammatory response in fish. Also, l-carnitine supplementation can relieve this bad condition. In conclusion, l-carnitine regulated Nrf2/Keap1 activation in vitro and in vivo and protected oxidized fish oil-induced inflammation response by inhibiting the NF-κB signaling pathway in Rhynchocypris lagowski Dybowski.

Oral immunization with recombinant Lactobacillus casei expressing flaB confers protection against Aeromonas veronii challenge in common carp, Cyprinus carpio.

Aeromonas veronii is an important type of gram-negative pathogen of human-livestock-aquatic animal and causes great economic losses in the aquaculture industry. Vaccination is an effective method of defence against A. veronii. There are many factors that restrict the use of vaccination, and the development of new oral vaccines is urgently needed. The selection of suitable antigens is of great significance for the development of aquaculture vaccines. Bacterial flagellin can specifically bind to TLR5 and induce the release of cytokines from the organism, which could be used in the development of vaccines. In this study, we constructed two recombinant Lactobacillus casei (L. casei) (surface-displayed or secretory) expressing the flaB of A. veronii and evaluated the effect of immune responses in common carp. The flaB gene (900 bp) of A. veronii was subcloned into the L. casei expression plasmids pPG-1 (surface-displayed) and pPG-2 (secretory). Western blot and immunofluorescence assays confirmed the expression of the recombinant flaB protein. Common carp immunized with Lc-pPG-1-flaB and Lc-pPG-2-flaB via oral administration route exhibited induction of antibody expression and innate immune responses. The results indicated that Lc-pPG-1-flaB and Lc-pPG-2-flaB can induce high levels of IgM, ACP, AKP, LZM and SOD activity in organisms, and Lc-pPG-1-flaB can induce even higher levels. The recombinant L. casei may effectively induce humoral immunity and increase the serum immunological index. Furthermore, leukocytes phagocytosis percentage and index of the recombinant L. casei were enhanced. The results of qRT-PCR showed that recombinant L. casei can significantly increase the expression of IL-10, IL-ß, IFN-γ and TNF-α in the tissues of immunized common carp, compared with control groups. Viable recombinant L. casei strains, which were delivered directly survived throughout the intestinal tract. Common carp that received Lc-pPG-1-flaB (66.7%) and Lc-pPG-2-flaB (53.3%) exhibited higher survival rates than the controls after challenge with the pathogen A. veronii. Our work indicated that Lc-pPG-1-flaB and Lc-pPG-2-flaB had beneficial effects on immune response and enhanced the disease resistance of common carp against A. veronii infection. The combination of flaB delivery and the Lactic acid bacteria (LAB) approach may be a promising method for the development of oral vaccines for treating A. veronii. In future research, we will focus on the colonization ability of LAB in the intestines and on the impact of these bacteria on intestinal flora.

Effect of sub-chronic exposure to selenium and Allium mongolicum Regel flavonoids on Channa argus: Bioaccumulation, oxidative stress, immune responses and immune-related signaling molecules.

Selenium (Se) is a micronutrient that becomes toxic when present at higher concentrations in fish tissues. Allium mongolicum Regel flavonoids (AMRF) have been documented to possess antioxidant, immunoenhancement and anti-inflammation properties. The aim of this study was to investigate the protective effects and potential mechanisms of dietary supplementation of AMRF and Se exposure on oxidative stress, immune responses and immune-related genes expression in Channa argus. A total of 480 C. argus were randomly divided into eight groups housed in twenty-four 200 L glass aquarium (3 tanks per group, 20 fish per tank). The fish were exposed for 56 days to waterborne Se at 0, 50, 100 and 200 µg/L and/or dietary AMRF at 40 mg/kg. The result indicated that AMRF exerted significant protective effects by preventing alterations in the levels of bioaccumulation, malondialdehyde, lysozyme, complement C3 and immunoglobulin M. AMRF also assists in the elevation of catalase and glutathione peroxidase in the liver and spleen while regulating the expression of immune-related genes including NF-κB p65, IκB-α, TNF-α, IL-1ß, IL-8, HSP70, HSP90, and glucocorticoid receptor after 56 days of Se exposure. Our results suggest that administration of AMRF (40 mg/kg) has the potential to combat Se toxicity in C. argus.

Effects of dietary Allium mongolicum Regel polysaccharide on growth, lipopolysaccharide-induced antioxidant responses and immune responses in Channa argus.

The present study evaluated the effects of dietary Allium mongolicum Regel polysaccharide (AMRP) on growth, lipopolysaccharide-induced antioxidant responses and immune responses in Channa argus. A basal diet was supplemented with AMRP at 0, 1, 1.5 or 2 g/kg feed for 56 days. After the 56 days feeding period, weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR) were significantly increased or decreased (P < 0.05) by dietary AMRP, with the highest WG, SGR and the minimum FCR occurring in 1.5 g/kg AMRP group. Furthermore, AMRP supplementation conferred significant protective effects against LPS challenge by preventing alterations in the levels of complements 3 (C3) and complements 4 (C4), lysozyme, superoxide dismutase (SOD), glutathione-S-transferase (GST), interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α) while regulating the expression of immune-related genes including heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), SOD, GST, IL-1 and TNF-α. Finally, AMRP supplementation significantly increased serum total protein, albumin and globulin concentrations and reduced mortality after LPS challenge. Taken together, our results suggest that the administration of AMRP could attenuate LPS-induced negative effects in C. argus, with 1.5 g/kg considered a suitable dose.

Oral Administration of Lactobacillus Casei Expressing Flagellin A Protein Confers Effective Protection against Aeromonas Veronii in Common Carp, Cyprinus Carpio.

Aeromonas veronii is a pathogen capable of infecting humans, livestock and aquatic animals, resulting in serious economic losses. In this study, two recombinant Lactobacillus casei expressing flagellin A (FlaA) of A. veronii, Lc-pPG-1-FlaA (surface-displayed) and Lc-pPG-2-FlaA (secretory) were constructed. The immune responses in fish administered with recombinant L. casei were evaluated. The two recombinant L. casei were orally administered to common carp, which stimulated high serum IgM and induced higher ACP, AKP, SOD and LYZ activity. Using qRT-PCR, the expression of IL-10, IL-8, IL-1ß, TNF-α and IFN-γ in the tissue of fish immunized with recombinant L. casei was significantly (p < 0.05) upregulated, which indicated that recombinant L. casei could activate the innate immune system to trigger the cell immune response and inflammatory response. Furthermore, recombinant L. casei was able to survive the intestinal environment and colonize in intestine mucosal. The study showed that after being challenged by A. veronii, fish administered with Lc-pPG-1-FlaA (70%) and Lc-pPG-2-FlaA (50%) had higher survival rates compared to Lc-pPG and PBS, indicating that recombinant L. casei might prevent A. veronii infection by activating the immune system to trigger immune responses. We demonstrated that flagellin as an antigen of vaccine, is acceptable for preventing A. veronii infection in fish. The recombinant L. casei expressing FlaA may be a novel mucosal vaccine for treating and controlling A. veronii.

Detection of floR gene and active efflux mechanism of Escherichia coli in Ningxia, China.

Florfenicol is an antibiotic, a fluorinated structural analogue of thiamphenicol and chloramphenicol, approved exclusively for use in Asia for aquaculture since the 1980's. Our study examined the prevalence of florfenicol resistance in bovine mastitis Escherichia coli isolates. A total of 245 E. coli isolates were collected from bovine mastitis in Ningxia Province, China between May 2016 to July 2017 and screened for florfenicol resistance gene, floR gene by PCR analysis. About 7.35% (15/245) of the isolates were positive for floR gene. Minimal inhibitory concentration (MIC) results showed that 9 isolates were susceptible strains and 6 isolates were highly resistant to florfenicol. HPLC (high performance liquid chromatography) results showed that the amounts of florfenicol was significantly improved in the presence of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) as an efflux pump inhibitor. This, therefore, indicates that the employment of florfenicol in conjunction with CCCP in future drug formulations should be considered.

Identification of a Newly Isolated Getah Virus in the China-Laos Border, China.

In this study, we isolated a virus strain (YN12031) from specimens of Armigeres subalbatus collected in the China-Laos border. BHK-21 cells infected with YN12031 exhibited an evident cytopathic effect (CPE) 32 h post-infection. The virus particles were spherical, 70 nm in diameter, and enveloped; they also featured surface fibers. Molecular genetic analysis revealed that YN12031 was closely related to alpha viruses such as Chikungunya virus and Sindbis virus, and located in the same clade as MM2021, the prototype of Getahvirus (GETV) isolated in Malaysia in 1955. Phylogenetic analysis of the E2 and capsid genes further revealed that YN12031 was located in the same clade as the Russian isolate LEIV/16275/Mag. Analysis of the homology of nucleotides and amino acids in the coding area and E2 gene demonstrated that the YN12031 isolated from the China-Laos border (tropical region) was related closest to the LEIV/16275/Mag isolate obtained in Russia (North frigid zone area) among other isolates studied. These results suggest that GETV can adapt to different geographical environments to propagate and evolve. Thus, strengthening the detection and monitoring of GETV and its related diseases is very crucial.

Modulatory role of L-carnitine against microcystin-LR-induced immunotoxicity and oxidative stress in common carp.

Microcystin-LR (MCLR), one of the most popular microcystins (MCs) found in many field water bodies around the world, poses great health risks to animals and humans. In the present study, healthy common carp (initial weight 24.8 ± 2.3 g) were randomly assigned to five groups. Group I was fed on normal diet as control. Group II was maintained on normal diet and received MCLR intraperitoneal injection (150 µg kg-1 BW). Common carp in groups III, IV, and V were daily pretreated with L-carnitine (LC) at doses of 0.5, 1.0, and 2.0 g kg-1 of the diet for 4 weeks prior to MCLR intraperitoneal injection. The results showed that MCLR alone led to a significant downregulation in immune response, including serum complement C3, lysozyme, and bactericidal activity. However, oxidative stress response: catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx), and lipid peroxidation (LPO) levels were significantly increased. Similarly, gene expressions of inflammatory IL-1ß, TNF-α, IFN I, and heat shock proteins (HSP70 and HSP90) were also upregulated after challenged with MCLR. However, LC pretreated group caused a significant elevation in immune response (C3, lysozyme, and bactericidal activity) and gene expressions of inflammatory IL-1ß, TNF-α, IFN I, and heat shock proteins (HSP70 and HSP90) after MCLR stress. Antioxidant activities (CAT, SOD, GSH, GPx, and LPO) were returned to background levels at 96 h after MCLR challenge. Strikingly, LC supplementation at 2.0 g kg-1 has been considered the optimum for common carp since it exhibited enhancement of immune response and antioxidant activity over the level 0.5 and 1.0 g kg-1, and even better than that of control level. It was concluded that LC as a functional feed additive significantly inhibited the progression of MCLR-induced immunotoxicity and oxidative stress in common carp.

Effects of Ala-Gln feeding strategies on growth, metabolism, and crowding stress resistance of juvenile Cyprinus carpio var. Jian.

The present study was conducted to evaluate the effects of different L-alanyl-l-glutamine (Ala-Gln) feeding strategies on the growth performance, metabolism and crowding stress resistance related parameters in juvenile Jian carp (Cyprinus carpio var. Jian) under crowded condition (80 g/L). Juvenile Jian carp (initial weight 26.1 ± 0.6 g) were distributed into five groups which fed with graded concentrations (0% or 1.0%) of Ala-Gln for eight weeks. Control group (I, 0/0) fed with control diet (0% Ala-Gln) throughout the feeding trial. The other four groups employed different control and experimental diet feeding strategies ranging from two weeks control diet fed and two weeks experimental diet (1% Ala-Gln) fed (II, 0/2) to eight weeks experimental diet fed (V, 4/4). Results revealed that Mean weight gain (MEG) under all different feeding strategies of Ala-Gln were significantly higher than that of the control group (p < 0.05), and MEG of group II (201.90%) was even higher than that of group IV (184.70%). Liver glycogen and blood total protein of groups II, III and V were significantly higher than that in groups I and IV (p < 0.05). The highest level of serum thyroxine (10.07 ng/ml), insulin-like growth factor-I (52.40 ng/ml) and insulin (9.73 µ IU/mL) were observed in group V. However, diet supplemented with Ala-Gln did not affect the levels of serum glucose, cortisol and catecholamine in fish. The mRNA expression of GR1a, GR1b and GR2 were also significantly changed in Ala-Gln supplementation groups compared with control group (p < 0.05). After fish intraperitoneally injected with virulent Aeromonas hydrophila, the fish survival rates were significantly increased in all Ala-Gln supplementation groups compared with control group (p < 0.05). Results from the present experiment showed the importance of dietary supplementation of Ala-Gln in benefaction of the growth performance, metabolism and crowding stress resistance in Jian carp breeding. The optimal feeding strategy was alternatively fed with control diet and then experimental diet at an interval of two weeks for juvenile Jian carp under crowded condition.

Enhancement of secondary metabolites from Bacillus Licheniformis XY-52 on immune response and expression of some immune-related genes in common carp, Cyprinus carpio.

This study was undertaken to isolate active secondary metabolites from immunostimulatory Bacillus Licheniformis XY-52 and evaluate their activities at 0%, 0.5%, 1.0%, and 2.0% doses supplementation with feed on immune response in common carp at weeks 1, 2, and 3. By applying chromatography techniques and successive recrystallization, two purified metabolites were obtained and identified by spectral data (mass spectrometry and nuclear magnetic resonance) as: Cyclo-(Phe-Tyr) and Cyclo-(Phe-Gly). The results revealed that humoral innate immune parameters (lysozyme activity, phagocytic activity and bactericidal activity) were significantly (P < 0.05) increased after feeding on the two active compounds-supplemented diet. Furthermore, administration of the two active compounds significantly (P < 0.05) up regulated IL-1ß, Type 1 IFN, IFN g2b, IL10 and TNF-α gene expression. Heat shock protein 70 (HSP70) gene expression was significantly (P < 0.05) lower as compared to control group at the end of trial. Common carp fed with the two compounds had higher survival rates (69.3%) compared to the controls (32.0%) after challenged with the pathogen, Aeromonas hydrophila. The present study indicates that the two isolated active compounds could positively influence immune response and enhance disease resistance of common carp against A. hydrophila infection.

Prevalence of 16S rRNA Methylase Gene rmtB Among Escherichia coli Isolated from Bovine Mastitis in Ningxia, China.

The aim of this study is to understand the prevalence and molecular characterization of 16S rRNA methylase gene, rmtB, among Escherichia coli strains isolated from bovine mastitis in China. A total of 245 E. coli isolates were collected from bovine mastitis in China between 2013 and 2014 and were screened for 16S rRNA methylase genes (armA, rmtA, rmtB, rmtC, rmtD, rmtE, and npmA) by polymerase chain reaction. About 5.3% (13/245) of the isolates carried the rmtB gene; the isolates were highly resistant to amikacin. Thirteen rmtB-positive strains were analyzed for the presence of extended-spectrum ß-lactamase genes (bla(TEM), bla(CTX-M), bla(OXA), and bla(SHV)). All the isolates harbored both bla(TEM-1) and bla(CTX-M-15) genes and two of the isolates were also positive for bla(OXA-1). Pulsed-field gel electrophoresis (PFGE) analysis indicated that the nine rmtB-positive strains belonging to ST10 from one farm showed the similar PFGE pattern, indicating a clonal expansion in this farm. S1-PFGE and Southern blotting showed that 12 isolates harbored the rmtB gene in plasmids of two different sizes (≈45 kb [n=10] and ≈48 kb [n=2]), while only 1 strain harbored the rmtB gene in the chromosome. These plasmids were transferable by conjugation studies, and two isolates from two respective farms carried the same size of plasmid, suggesting that the horizontal transmission of plasmids also contributed to the spread of rmtB gene. This is the first report of prevalence of the 16S rRNA methylase gene rmtB among E. coli isolated from bovine mastitis in China, and rmtB-carrying E. coli may pose a threat to the treatment of bovine mastitis.

Incidence and clinical analysis of asymptomatic intracranial hemorrhage in neonates with cerebral hypoxic-ischaemic risk based on multisequence MR images.

The incidence and clinical distribution of intracranial haemorrhage (ICH) in neonates at risk of cerebral hypoxia-ischaemia have not been reported in specific studies. Based on conventional magnetic resonance imaging (MRI) versus susceptibility weighted imaging (SWI), this study aimed to analyse the occurrence of asymptomatic ICH in newborns with or without risk of cerebral hypoxia-ischaemia and to accumulate objective data for clinical evaluations of high-risk neonates and corresponding response strategies. 317 newborns were included. MRI revealed that the overall incidence of ICH was 59.31%. The most common subtype was intracranial extracerebral haemorrhage (ICECH) which included subarachnoid haemorrhage (SAH) and subdural haemorrhage (SDH). ICECH accounted for 92.02% of ICH. The positive detection rate of ICECH by SWI was significantly higher than that by T1WI. The incidence of total ICH, ICECH and SAH was greater among children who were delivered vaginally than among those who underwent caesarean delivery. Asymptomatic neonatal ICH may be a common complication of the neonatal birth process, and SWI may improve the detection rate. Transvaginal delivery and a weight greater than 2500 g were associated with a high incidence of ICECH in neonates. The impact of neonatal cerebral hypoxia-ischaemia risk factors on the occurrence of asymptomatic ICH may be negligible.

Identification of compounds from Paris polyphylla (ChongLou) active against Dactylogyrus intermedius.

The present study was designated to ascertain the anthelmintic activity of the rhizomes of Paris polyphylla and to isolate and characterize the active constituents. The methanol extract from rhizomes of P. polyphylla showed significant anthelmintic activity against Dactylogyrus intermedius with the median effective concentration (EC50) 22.5 mg L(-1). Based on this finding, the methanol extract was fractionated by silica gel column chromatography in a bioassay-guided fractionation yielding 2 bioactive compounds, the structures of these compounds were elucidated as formosanin C and polyphyllin VII. The in vivo tests revealed that formosanin C and polyphyllin VII were significantly effective against D. intermedius with EC50 values of 0.6 and 1.2 mg L(-1), respectively. The acute toxicities (LC50) of formosanin C and polyphyllin VII for grass carp were 2.8 and 2.9 mg L(-1), respectively. The overall results provide important information for the potential application of formosanin C and polyphyllin VII in the therapy of serious infection caused by D. intermedius.

[Migratory properties of vascular smooth muscle cells on extracellular matrix: a study on inverted coverslip migration assay].

Migration of vascular smooth muscle cells (VSMCs) is involved in vascular development and various vascular diseases; however, the molecular mechanisms of VSMC migration remain unclear. In this study, we established an inverted coverslip migration assay to study the migratory properties of cultured VSMCs on extracellular matrix. Pulmonary arterial smooth muscle cells (PASMCs) from rats were cultured and identified by immunocytochemistry. Each coverslip with a confluent monolayer of PASMCs was inverted to a larger coverslip which was coated with phosphate buffered saline (PBS, as a control), poly-D-lysine hydrobromide (PDL), laminin or Matrigel. After 24 h of migration over the larger coverslip, PASMCs were fixed, and reliably quantified. The roles and mechanisms of extracellular matrix in PASMC migration were further studied by wound-healing assay and immunocytochemistry. The results showed that: (1) The purity of the cultured PASMCs was (97 ± 3)%. (2) The number of PASMCs on laminin or Matrigel migrating out from the inverted coverslip was significantly increased compared with that on PBS or PDL, and the migratory distance of PASMCs on laminin or Matrigel was significantly farther than that on PBS or PDL. (3) The motility of PASMCs on laminin or Matrigel was significantly higher than that on PBS at 8 h, 12 h and 24 h after wounding, respectively. (4) F-actin staining showed that F-actin was congregated along the brim of the migrating cells from the inverted coverslip, and vinculin (a cell marker of focal adhesion) staining showed that the distribution of vinculin in PASMCs plated on laminin or Matrigel was significantly lower than that on PBS or PDL. These results suggest that the inverted coverslip migration assay is suitable to study VSMC migration, and laminin and Matrigel substrates may promote VSMC migration through inhibiting the formation of focal adhesion and regulating the cytoskeletal proteins.