muramidase and Gram-Negative-Bacterial-Infections

Topics: Aeromonas caviae; Animals; Carps; Fish Diseases; Gram-Negative Bacterial Infections; Immunoglobulin M; Interleukin-10; Mammals; Muramidase; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Vaccines, Inactivated

Several studies have looked into the use of basil, Ocimum basilicum (L.) in aquaculture as a dietary additive; however, more research is needed to see the possibility of it's including in nanocarriers in aquafeeds. An experiment was undertaken to highlight the efficacy chitosan-Ocimum basilicum nanocomposite (COBN), for the first time, on Nile tilapia (Oreochromis niloticus) growth, stress and antioxidant status, immune-related parameters, and gene expression. For 60 days, fish (average weight: 23.55 ± 0.08 g) were fed diets provided with different concentrations of COBN (g/kg): 0 g [COBN0], 1 g [COBN1], 2 g [COBN2], and 3 g [COBN3], where COBN0 was kept as control diet. Following the trial, the fish were challenged with pathogenic bacteria (Aeromonas sobria) and yeast (Candida albicans) infection. In comparison to the control (COBN0), a notable increase in growth parameters (weight gain, feed intake, and specific growth rate) and intestinal morphometric indices (average intestinal goblet cells count, villous width, and length) in all COBN groups was observed, where COBN2 and COBN3 groups had the highest values. The COBN diets significantly (p < 0.05) declined levels of serum triglycerides, glucose, cholesterol, and hepatic malondialdehyde. Moreover, the higher levels of serum biochemical biomarkers (growth hormone, total protein, globulin, and albumin), immunological parameters (phagocytic activity%, nitric oxide, and lysozyme), and hepatic antioxidant parameters (superoxide dismutase, total antioxidant capacity, and glutathione peroxidase) were obvious in the COBN2 and COBN3 groups followed by COBN1. The immune-antioxidant genes (TNF-α, IL-10, IL-1β, TGF-β, GPx, and SOD) were found to be considerably up-regulated in all COBN groups (COBN2 and COBN3 followed by COBN1). Fifteen days post-challenge with A. sobria and C. albicans, the highest survival rate was recorded in the COBN2 group (83.33 and 91.67%) followed by the COBN3 group (75 and 83.33%), respectively. The findings showed that a dietary intervention with COBN can promote growth, intestinal architecture, immunity, and antioxidant markers as well as protect O. niloticus against A. sobria and C. albicans infection. As a result, the COBN at a dose of 2 g/kg could be used as a food additive for the sustainable aquaculture industry.

Topics: Albumins; Animal Feed; Animals; Antioxidants; Chitosan; Cichlids; Diet; Dietary Supplements; Fish Diseases; Food Additives; Gene Expression; Glucose; Glutathione Peroxidase; Gram-Negative Bacterial Infections; Growth Hormone; Head Kidney; Interleukin-10; Malondialdehyde; Muramidase; Nanocomposites; Nitric Oxide; Ocimum basilicum; Superoxide Dismutase; Transforming Growth Factor beta; Triglycerides; Tumor Necrosis Factor-alpha

Medicinal herbs are used for growth promotion, disease control and other health benefits in aquaculture industry. Here, we examined the effect of dietary laurel-leaf cistus (Cistus laurifolius) ethanolic extract on growth performance, digestive enzyme activity, haematological profile and nonspecific immune responses in common carp (Cyprinus carpio). In addition, resistance against Aeromonas hydrophila infection was examined. Common carp was fed diets containing 0 (Control), 0.1 (CL0.1), 0.5 (CL0.5) and 1 (CL1) g kg

Topics: Aeromonas hydrophila; Amylases; Animals; Blood Cell Count; Carps; Cistus; Cytokines; Ethanol; Fish Diseases; Gram-Negative Bacterial Infections; Head Kidney; Immunity, Innate; Immunologic Factors; Lipase; Muramidase; Plant Extracts; Plant Leaves; Solvents; Superoxides; Trypsin

The waste recycling of lemon peel, as a functional feed additive in aquafeed was evaluated by estimating the effects of fermented lemon peel (FLP) supplementation in diet on growth performance, innate immune responses, and susceptibility to Photobacterium damselae of grouper, Epinephelus coioides. A basal diet was added FLP at 0%, 1%, 3%, and 5%. Four tested diets were each fed to juvenile grouper (initial weight: 15.89 ± 0.10 g, triplicate groups) in a recirculation rearing system for eight weeks. Fish fed diets with 0%-3% FLP exhibited higher (p < 0.05) final weight, weight gain, and feed efficiency than fish fed the 5% FLP-diet. After challenge test, fish fed the 3% FLP-diet appeared the lowest mortality, followed by fish fed the 1% FLP-diet, and lowest in fish fed 0% and 5% FLP-diets. Plasma lysozyme activities were higher in fish fed diets with FLP than in fish fed the FLP-free control diet before challenge test. After challenge, fish fed diets with 1% and 3% FLP showed highest lysozyme activities, followed by fish fed the diet with 5% FLP, and lowest in fish fed the control diet. Hepatic malondialdehyde content was higher in fish fed the control diet than in fish fed diets with 1%-3% FLP. Results found that diets supplemented with 1%-3% fermented lemon peel can enhance lysozyme activity and resistance to pathogen P. damselae of grouper.

Topics: Animals; Citrus; Dietary Supplements; Disease Susceptibility; Fermentation; Fish Diseases; Fruit; Gram-Negative Bacterial Infections; Liver; Malondialdehyde; Muramidase; Perciformes; Photobacterium

This study investigated the immunomodulatory effects of Sargassum polycystum extract administration in rainbow trout (Oncorhynchus mykiss). S. polycystum methanolic extract was administered orally using feeding needles to individual rainbow trout at the dose of 0 (control), 1 (S1), 3 (S3) and 5 (S5) mg/100 μl/per fish twice a day for 7 days. On 1st, 5th, 3rd and 7th day, blood and tissues were collected from the fish and changes in humoral immune responses and immune-related gene expressions were determined. The result of oxidative radical production showed no difference during early stage of the experiment and was lately decreased (P < 0.05). Lysozyme activity increased on 3rd and 7th day of the study in S5 fish group and on 5th day in S3 group compared to control (P < 0.05). Myeloperoxidase activity had an increased level on the 1st and 3rd day in S1, S5 and S5 fish groups, respectively. IL-1β gene was significantly up-regulated in kidney and intestine in all experimental groups (except on the 1st day, in the intestine of S5 fish group) compared to control (P < 0.05). IL-8 gene expression was elevated on 1st and 3rd day in kidney of all experimental fish groups. IL-6 transcript enhanced in a dose-dependent manner on 3rd and 7th day. IL-10 and IL-12 genes were also up-regulated. Survival in all treated fish groups challenged with Aeromonas hydrophila was significantly increased compared to that of control. The highest survival rate was recorded in S5 fish group (83.65%) followed by S3 fish group (82.62%). Our results suggest that S. polycystum aqueous methanolic extract is an effective immunostimulant and provide protection against A. hydrophila infection in rainbow trout at a dose of 3-10 mg/20 g body weight/day.

Topics: Adjuvants, Immunologic; Administration, Oral; Aeromonas hydrophila; Animals; Complex Mixtures; Cytokines; Fish Diseases; Gram-Negative Bacterial Infections; Muramidase; Oncorhynchus mykiss; Peroxidase; Sargassum

The effect of diet enriched with dried lemon (Citrus limon) peel was fed to Labeo rohita at three different levels (0, 1, 2.5, and 5 g kg

Topics: Aeromonas; Animals; Antioxidants; Catalase; Citrus; Complement C3; Cyprinidae; Cytokines; Diet; Fish Diseases; Fish Proteins; Fruit; Glutathione; Glutathione Peroxidase; Gram-Negative Bacterial Infections; Head Kidney; Immunoglobulin M; Muramidase; Superoxide Dismutase

European seabass (Dicentrarchus labrax) production is often hampered by bacterial infections such as photobacteriosis caused by Photobacterium damselae subsp. piscicida (Phdp). Since diet can impact fish immunity, this work investigated the effect of dietary supplementation of 5% Gracilaria sp. aqueous extract (GRA) on seabass antioxidant capacity and resistance against Phdp. After infection, mortality was delayed in fish fed GRA, which also revealed increased lysozyme activity levels, as well as decreased lipid peroxidation, suggesting higher antioxidant capacity than in fish fed a control diet. Dietary GRA induced a down-regulation of hepatic stress-responsive heat shock proteins (grp-78, grp-170, grp-94, grp-75), while bacterial infection caused a down-regulation in antioxidant genes (prdx4 and mn-sod). Diet and infection interaction down-regulated the transcription levels of genes associated with oxidative stress response (prdx5 and gpx4) in liver. In head-kidney, GRA led to an up-regulation of genes associated with inflammation (il34, ccr9, cd33) and a down-regulation of genes related to cytokine signalling (mif, il1b, defb, a2m, myd88). Additionally, bacterial infection up-regulated immunoglobulins production (IgMs) and down-regulated the transcription of the antimicrobial peptide leap2 in head kidney. Overall, we found that GRA supplementation modulated seabass resistance to Phdp infection.

Topics: Animal Feed; Animals; Antimicrobial Cationic Peptides; Aquaculture; Bass; Blood Glucose; Cytokines; Dietary Supplements; Disease Resistance; Fish Diseases; Gene Expression Regulation; Gracilaria; Gram-Negative Bacterial Infections; Head Kidney; Heat-Shock Proteins; Immunoglobulin M; Lipid Peroxidation; Liver; Muramidase; Photobacterium; Triglycerides

The effects of dietary Bacillus coagulans (MTCC 9872), Bacillus licheniformis (MTCC 6824) and Paenibacillus polymyxa (MTCC 122) supplementation on growth performance, non-specific immunity and protection against Aeromonas hydrophila infection were evaluated in common carp, Cyprinus carpio fry. Laboratory maintained B. coagulans, B. licheniformis and P. polymyxa were used to study antagonistic activity against fish pathogenic bacteria by agar well diffusion assay. Healthy fish fry were challenged by this bacterium for determination of its safety. Fish were fed for 80 days with control basal diet (B0) and experimental diets containing B. coagulans (B1), B. licheniformis (B2) and P. polymyxa (B3) at 10(9) CFU/g diet. Fish fry (mean weight 0.329 ± 0.01 g) were fed these diets and growth performance, various non-specific immune parameters and disease resistance study were conducted at 80 days post-feeding. The antagonism study showed inhibition zone against A. hydrophila and Vibrio harveyi. All the probiotic bacterial strains were harmless to fish fry as neither mortality nor morbidities were observed of the challenge. The growth-promoting influences of probiotic supplemented dietary treatments were observed with fish fry and the optimum survival, growth and feed utilization were obtained with P. polymyxa (B3) supplemented diet. Study of different non-specific innate immunological parameters viz. lysozyme activity, respiratory burst assay and myeloperoxidase content showed significant (p < 0.05) higher values in fish fry fed B3 diet at 10(9) CFU/g. The challenge test showed dietary supplementation of B. coagulans, B. licheniformis and P. polymyxa significantly (p < 0.05) enhanced the resistance of fish fry against bacterial challenge. These results collectively suggests that P. polymyxa is a potential probiotic species and can be used in aquaculture to improve growth, feed utilization, non-specific immune responses and disease resistance of fry common carp, C. carpio.

Topics: Adjuvants, Immunologic; Analysis of Variance; Animals; Aquaculture; Bacillus; Carps; Dietary Supplements; Disease Resistance; Disk Diffusion Antimicrobial Tests; Dose-Response Relationship, Drug; Fish Diseases; Gram-Negative Bacterial Infections; Muramidase; Peroxidase; Probiotics; Respiratory Burst

A 60-day feeding trial was conducted to study the immuno-protective effect of microbial levan on Labeo rohita juveniles challenged with Aeromonas hydrophila. Six purified diets were prepared with different levels of microbial levan: control (no levan), T1 (Basal + 0.25%), T2 (Basal + 0.50%), T3 (Basal + 0.75%), T4 (Basal + 1%) and T5 (Basal + 1.25%), fed to six groups of fish in triplicate. Among the treatment groups the haemoglobin content and total leucocyte count were increased with a dietary supplementation of levan at 1% or more. An increasing trend for total erythrocyte count was observed with increasing level of dietary levan. Lower levan-supplemented groups showed a higher albumin/globulin ratio. As the levan supplementation was increased, there was a gradual increase in serum lysozyme activity and respiratory burst activity [nitroblue tetrazolium (NBT) assay] reduction values. The highest lysozyme activity and NBT were observed in the T5 group although this was similar to the T4 group (P > 0.05). No significant histo-architectural changes were associated with dietary levan levels. After challenge with A. hydrophila, moderately degenerated hepatocytes, oedema and leucocytic infiltration in parenchymatous tissues, and extensive haemorrhage and haemosiderosis in the kidney were observed in the control group. However, the T5 group supplemented with 1.25% levan showed infiltrating leucocytes in the liver while the kidney showed only moderate degeneration of renal tubules. The relative survival per cent of juveniles after challenge with A. hydrophila was the highest in the T5 group followed by T4. This suggests that microbial levan at 1.25% can be used as dietary immunostimulant for L. rohita juveniles.

Topics: Aeromonas hydrophila; Animals; Cyprinidae; Diet; Fish Diseases; Fructans; Gram-Negative Bacterial Infections; Hepatocytes; Immunity, Innate; Kidney; Liver; Muramidase; Probiotics; Respiratory Burst; Survival Analysis

Clostridium butyrium CB2 isolated from chickens was tested as a potential fish probiotic in the Chinese drum, Miichthys miiuy. Fish were fed live (CB), dead CB2 (D-CB) cells (10(8) cells g(-1)) or spent culture supernatant (SCS), for 30 days and challenged with Vibrio anguillarum or Aeromonas hydrophila. Survival was higher in both the CB and the CB-D fed groups, but the SCS group was not significantly different from the control. After feeding live or dead CB2 cells, there was increase in phagocytic activity of the head kidney macrophages, the lysozyme activity of serum and gut mucosa and immunoglobulin (Ig) level. The SCS group showed no obvious change in immune parameters. The results suggest that live or dead Clostridium butyrium CB2 has an immunomodulatory effect on fish.

Topics: Administration, Oral; Aeromonas hydrophila; Animals; Chickens; Clostridium; Culture Media; Gram-Negative Bacterial Infections; Immunity, Innate; Immunoglobulins; Immunologic Factors; Intestinal Mucosa; Macrophages; Microbial Sensitivity Tests; Muramidase; Perciformes; Probiotics; Survival Analysis; Vibrio; Vibrio Infections

A preliminary study with 60 days feeding was conducted to study the immunomodulatory role of different immunostimulants like beta-carotene, omega-3 fatty acid and yeast-RNA in Catla catla fingerlings. Two hundred and sixty four fingerlings were randomly distributed into eight treatment groups with each of three replicates. Eight isonitrogenous (crude protein 34.12-35.40%) and isocaloric (458.41-461.48 kcal/100g) purified diets were prepared with graded level of beta-carotene, omega-3 fatty acid and yeast-RNA viz., Control (basal diet), T1 (Basal + 1% omega-3 fatty acid), T2 (Basal + 3% omega-3 fatty acid), T3 (Basal + beta-carotene), T4 (T1 + beta-carotene), T5 (T2 + beta-carotene), T6 (Basal + 0.4% yeast-RNA) and T7 (Basal + 0.8% yeast-RNA). The immunomodulatory effects of dietary immunostimulants were studied in terms of respiratory burst activity (NBT) of blood phagocytes, total leukocyte count, serum total protein, serum globulin, A/G ratio (A/G) and serum lysozyme activity. The respiratory burst activity of T7 group was significantly higher (p<0.05) than the other groups. Haemoglobin content, total erythrocyte count and serum albumin content did not vary among the treatment groups, whereas total leukocyte count, serum globulin content and serum lysozyme activity were found to be highest in T7 group. Relative survival percent after challenge with Aeromonas hydrophila was also highest in T7 (88.88%) group followed by T6 (75.06%) and T4 (66.66%) and the lowest in T2 group. It was observed that total leucocyte count, NBT and lysozyme activity of T2 group fed with high omega-3 fatty acid (3%) was less than (p<0.05) its lower counterparts T1 (1%) and control group. Based on the results of the present study, it concludes that supplementation of yeast-RNA at 0.8% registered higher immunological responses in C. catla juveniles. It is also observed that higher supplementation of omega-3 fatty acid (3%) in the diet causes immunosuppression in C. catla juveniles.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animal Feed; Animals; beta Carotene; Blood Proteins; Cyprinidae; Dietary Supplements; Eating; Erythrocyte Count; Fatty Acids, Omega-3; Fish Diseases; Gram-Negative Bacterial Infections; Hemoglobins; Immunity, Innate; Leukocyte Count; Muramidase; Random Allocation; Respiratory Burst; RNA, Fungal

The effects of including bovine lactoferrin (Lf) in the diet of the Asian catfish (Clarias batrachus) on specific and non-specific immunity as well as disease resistance were investigated. The catfish were fed four different diets for 2 weeks: a commercial diet as control and the same diet supplemented with 50, 100 and 200mg bovine Lf/kg feed. After 1 and 2 weeks, serum bacterial agglutination titre against Aeromonas hydrophila as a measure of specific immunity; natural serum haemolysin titre, lysozyme activity and oxidative radical production by neutrophils as a measure of non-specific immunity as well as disease resistance against A. hydrophila challenge to vaccinated and non-vaccinated animals were evaluated. The results showed that Lf supplements, particularly at 100mg level, significantly (P<0.05) enhanced serum lysozyme level, oxidative radical production and level of protection against A. hydrophila challenge in non-vaccinated animals irrespective of length of exposure. The specific immunity was not influenced by Lf feeding as evidenced from the bacterial agglutination titre and level of protection in vaccinated animals. As Lf feeding at 100mg/kg for 1 week is able to enhance the non-specific immunity and disease resistance of catfish efficiently, these results support the possible use of Lf as an immunostimulant for farmed catfish.

Topics: Aeromonas hydrophila; Animal Feed; Animals; Antibodies; Bacterial Vaccines; Catfishes; Cattle; Diet; Disease Susceptibility; Dose-Response Relationship, Drug; Fish Diseases; Gram-Negative Bacterial Infections; Hemolysin Proteins; Lactoferrin; Muramidase

Topics: Animal Feed; Animals; Antioxidants; Carps; Diet; Dietary Supplements; Disease Resistance; Gram-Negative Bacterial Infections; Immunity, Mucosal; Immunoglobulin M; Muramidase; Plant Leaves; RNA, Messenger; Superoxide Dismutase

Common carp (Cyprinus carpio), a valuable aquaculture species susceptible to various infections, requires effective immune enhancement strategies. This study investigates the immunomodulatory effects of orally administered terpenoids and phenol fraction (TPF) from Padina gymnospora in C. carpio, focusing on stimulation of nonspecific immune response, immune gene expression, and protection against experimental infection. P. gymnospora is a brown seaweed species known for its bioactive compounds and medicinal properties. TPF was extracted using the Harborne fractionation method, and the presence of terpenoids and phenol compounds was confirmed by qualitative analysis and high-performance thin layer chromatography (HPTLC). TPF was administered orally in different doses to carp. Nonspecific immune responses were evaluated by measuring cellular ROS, RNI, and peroxidase production. The expression of immune genes (lysozyme and interleukin-1β) was assessed by reverse transcriptase PCR. Furthermore, the protective efficacy of TPF was determined by infecting carp with a virulent pathogen, Aeromonas hydrophila, and monitoring mortality rates and disease symptoms. The results demonstrate that oral TPF administration significantly enhances nonspecific immune responses, with increased ROS, RNI, and peroxidase production, indicating improved immune function. Expression levels of lysozyme and interleukin-1β were upregulated, suggesting immune system activation. Moreover, TPF exhibited significant protection against experimental infection, with lower mortality rates compared to the control group. These findings highlight TPF's potential as an effective immunostimulatory agent, enhancing immune responses and providing infection protection in carp. In conclusion, oral TPF administration stimulates nonspecific immune responses, modulates immune gene expression, and confers protection against experimental infection in carp, displaying its potential for enhancing immune responses and disease resistance in aquaculture species, and contributing to sustainable fish health management.

Topics: Administration, Oral; Aeromonas hydrophila; Animals; Carps; Disease Resistance; Fish Diseases; Gram-Negative Bacterial Infections; Interleukin-1beta; Muramidase; Peroxidases; Phenol; Reactive Oxygen Species; Terpenes

Aquatic food is becoming an important food source that provides micronutrients to human beings. The decline of wild aquatic animals makes aquaculture become increasingly important to play this role. However, infectious diseases, especially bacterial infection, represent severe threat to aquaculture, which causes huge economic loss. Meanwhile, strategies in managing bacterial infection in an antibiotic-independent way are still lacking. In this study, we monitor the metabolomic shift of crucian carp upon

Topics: Aeromonas hydrophila; Animals; Carps; Fish Diseases; Fructose; Gram-Negative Bacterial Infections; Muramidase

Lysozyme (Lyz) is an alkaline enzyme that hydrolyzes mucopolysaccharides in bacteria and is highly conserved vertebrates and invertebrates. In this study, a c-type lysozyme gene (named ToLyzC) from the obscure puffer Takifugu obscurus was cloned and characterized. The full-length cDNA of ToLyzC was 432 bp, encoding 143 amino acids, with a predicted molecular mass of 16.2 kDa and a theoretical pI of 8.86. The depicted protein sequence contained a LYZ1 domain from 16 to 142 amino acids, seven conserved cysteine residues. Phylogenetic analysis indicated that ToLyzC clustered with Lyzs from other teleost fishes. Quantitative real-time PCR analysis revealed that ToLyzC mRNA was mainly expressed in the liver. The transcript level of ToLyzC gene was significantly upregulated after Staphylococcus aureus and Vibrio harveyi challenge. The optimal pH and temperature of recombinant ToLyzC protein (rToLyzC) lytic activity was detected to be 7.5 and 35 °C, respectively. rToLyzC exhibited significant antibacterial and bacterial binding activities against S. aureus, Aeromonas hydrophila, V. harveyi, and Edwardsiella tarda at different time points. In addition, the morphological changes of V. harveyi cells treated with rToLyzC were observed under scanning electron microscope, which further confirmed the antibacterial and bacteriolytic activity of rToLyzC. Taken together, our current study indicated that ToLyzC is involved in the immune response to bacterial infection in obscure puffers.

Topics: Amino Acids; Animals; Anti-Bacterial Agents; Gram-Negative Bacterial Infections; Muramidase; Phylogeny; Staphylococcus aureus; Takifugu

A pseudotuberculosis pathogen, Photobacterium damselae subsp. piscicida (Pdp), has caused enormous economic damage to yellowtail aquaculture in Japan. The Ivy gene has been discovered in plasmid of Pdp, and it has been proposed that it may help bacteria evade lysozyme-mediated lysis during interaction with an animal host. However, the lysozyme-inhibiting activity of Pdp-derived Ivy (Ivy-Pdp) is unknown, and it is unclear whether it acts as a virulence factor for host biophylaxis. In this study, the inhibitory effect of Ivy-Pdp on lysozyme was evaluated by expressing and purifying the recombinant Ivy-Pdp protein (rIvy-Pdp). The rIvy-Pdp protein inhibited hen egg white lysozyme activity in an rIvy-Pdp-concentration-dependent manner, and its inhibitory effect was similar under different temperature and pH conditions. The serum and skin mucus of the yellowtail (which is the host species of Pdp), Japanese flounder, and Nile tilapia showed bacteriolytic activity. In contrast, the addition of rIvy-Pdp inhibited the lytic activity in the serum of these fish species. In particular, it significantly inhibited lytic activity in the serum and skin mucus of Nile tilapia. On the basis of these results, we suggest that Ivy-Pdp is a temperature- and pH-stable lysozyme inhibitor. Additionally, Ivy-Pdp inhibited the lytic activity of lysozyme, which is involved in host biophylaxis. In summary, we inferred that Ivy-Pdp is an important factor that diminishes the sterilization ability of C-type lysozyme when Pdp infects the host.

Topics: Animals; Aquaculture; Fish Diseases; Gram-Negative Bacterial Infections; Muramidase; Perciformes; Photobacterium

Aeromonas veronii AvX005 is a pathogenic bacterium with high toxicity to grass carp (Ctenopharyngodon idellus). The expression levels of g-type (goose-type lysozyme, Lys-g) and c-type lysozyme (chicken-type lysozyme, Lys-c) in the spleen of grass carp infected with AvX005 were significantly increased by approximately 4.5 times and 27 times, respectively. The recombinant proteins rLys-g and rLys-c produced in a recombinant expression system of Escherichia coli showed significant antibacterial activity against the pathogenic bacteria AvX005. A challenge test was conducted after rLys-g and rLys-c were expressed in grass carp L8824 liver cells, and compared with the survival rate of the control cells (46.3%), the survival rate of the experimental cells (77.6% for rLys-g and 68.6% for rLys-c) was significantly increased. Grass carp were infected with AvX005 on the second day after delivering pcDNA3.1-lys-g and pcDNA-lys-c with the Quil A/cholesterol/DDA/Carbopol (QCDC) adjuvant, and both pcDNA3.1-lys-g and pcDNA-lys-c provided 70% relative protection for grass carp. The activity of lysozyme and alkaline phosphatase in the serum of grass carp was significantly increased after injection of DNA. The expression of the immune factors IgM, C3 and IL8 in the kidney was upregulated to varying degrees for pcDNA3.1-lys-g and immune factors C3 and IgM was upregulated for pcDNA-lys-c. The results indicated that pcDNA3.1-lys-g and pcDNA-lys-c may be used as immunostimulants to protect grass carp from the pathogenic bacterium AvX005.

Topics: Acrylic Resins; Adjuvants, Immunologic; Aeromonas hydrophila; Aeromonas veronii; Animals; Carps; Cholesterol; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Immunoglobulin M; Muramidase; Quillaja Saponins

Natural products have gained considerable attention for improving fish growth performance and immunity to enhance disease resistance. This study evaluated the effect of dandelion polysaccharides (DP) on skin mucosal immune parameters, immune-related gene expression, and susceptibility to pathogen challenge in the Common carp Cyprinus carpio. Diets containing four different concentrations of DP (g Kg

Topics: Alkaline Phosphatase; Animal Feed; Animals; Antioxidants; Biological Products; Carps; Catalase; Claudin-3; Cytokines; Diet; Dietary Supplements; Fish Diseases; Food Additives; Gram-Negative Bacterial Infections; Interleukin-10; Malondialdehyde; Muramidase; Occludin; Polysaccharides; Superoxide Dismutase; Taraxacum; Tumor Necrosis Factor-alpha

Disease problems will seriously restrict the sustainable development of aquaculture, and the environmental-friendly prevention strategies are urgently needed. Probiotics and quorum-quenching enzyme are innovative strategies to control bacterial diseases. Firstly, the bacteriostatic activity of Bacillus subtilis wt55 strain and quenching enzyme AiiO-AIO6 on the growth of Aeromonas veronii were tested in vitro, and the results showed wt55 inhibit the growth of A. veronii, but AiiO-AIO6 did not. Then, the synergistic effects of simple combination of B. subtilis wt55 and AiiO-AIO6 were evaluated next. The results showed this combination could improve the survival rate and significantly reduce the number of invasive A. veronii in gut after challenge compared to the other groups, corresponding to the lower intestinal alkaline phosphatase activity. One of its effect mechanisms is the combination could inhibit the growth of A. veronii in vitro; the other is direct immersion of germ-free zebrafish proved AiiO-AIO6 did not directly regulate the innate immune response of the host, but wt55 did it, and the simple combination group could significantly reduce the expression of nuclear factor kappa-B (NF-κB) and proinflammatory cytokine interleukin-1β (IL-1β), increase the expression of lysozyme gene; and the third is intestinal microbiota also plays a regulatory role: the gut microbiota from combination group could significantly inhibit the expression of IL-1β and NF-κB, and increased the expression of transforming growth factor-β (TGF-β) and lysozyme. Given the effectiveness of this simple combination, a B. subtilis quorum-quenching recombinant expression strain in which AiiO-AIO6 was surface displayed on the spores and secreted by vegetative cells was built. The results showed that the survival rate after challenge was lower than that of the group treated with AiiO-AIO6 or wt55 alone, and the expression of proinflammatory cytokine IL-1β and NF-κB were significantly higher. Our study demonstrated the effectiveness of B. subtilis and AiiO-AIO6 simple combination and established an efficient B. subtilis expression system.

Topics: Aeromonas veronii; Alkaline Phosphatase; Animals; Bacillus subtilis; Fish Diseases; Gram-Negative Bacterial Infections; Interleukin-1beta; Muramidase; NF-kappa B; Transforming Growth Factor beta; Transforming Growth Factors; Zebrafish

The present study was conducted to evaluate the immunostimulatory effect of tea leaf extract (Camellia sinensis) on Labeo rohita and its resistance against Aeromonas hydrophila infection. The ethanolic extract of green tea (GTEE) was found to be the most potent as compared to other solvent extract which was used for further study. It was used to evaluate immune-biochemical response of L. rohita fingerlings, fed with tea leaf extract (control- 0.0%, 0.2% (T1), 0.4% (T2), 0.8% (T3) and 1% (T4) of GTEE kg

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Antioxidants; Blood Proteins; Cyprinidae; Fish Diseases; Gene Expression Profiling; Glucose; Gram-Negative Bacterial Infections; Immunity; Interleukin-10; Muramidase; Peroxidase; Plant Extracts; Solvents; Tea

An eight-week feeding trial was performed to assess the effect of different dietary levels (0, 0.5, 1.0, and 1.5%) of β-glucan (sourced from Saccharomyces cerevisiae) on growth, survival, immunological parameters (immune gene expression, lysozyme, and antiprotease), total antioxidant status, thermal tolerance, and disease resistance of Tor putitora fry. Feeding of moderate doses (0.5 and 1.0%) of β-glucan significantly improved survival but not weight gain percentage as compared to that received unsupplemented control and highest dose (1.5%) of glucan. Supplementation of β-glucan in diets differentially influenced the mRNA expression of cytokine and other immune genes. For instance, transcripts of cytokines such as tnf-α and il-1β were significantly upregulated, while ifn-γ and il-10 were unaffected by β-glucan intake. Also, the relative mRNA expression of tlr-5 and hepcidin1 along with lysozyme and antiprotease activities were remained largely unchanged by dietary glucan administration. In contrast, β-glucan induced mRNA expression of defensin1 and c3 while decreased the transcript level of mhc-1. On the other hand, dietary inclusion of β-glucan markedly improved total antioxidant levels and extended the thermal tolerance limits at both the ends, as shown by increased CT

Topics: Aeromonas salmonicida; Animal Feed; Animals; Antioxidants; beta-Glucans; Cyprinidae; Disease Resistance; Fish Diseases; Gene Expression; Gram-Negative Bacterial Infections; Muramidase; Protease Inhibitors; RNA, Messenger

Vaccination is the most effective, safe, and environmentally friendly method to prevent the outbreak of Photobacterium damselae subsp. piscicida (Phdp), a dangerous pathogen in aquaculture worldwide. Here, recombinant proteins of catalase, superoxide dismutase, isocitrate dehydrogenase, fructose 1,6-bisphosphate aldolase (Fba), and a mixture of all four proteins were investigated for their immunoprotective effects against photobacteriosis in Asian sea bass (Lates calcarifer). After immunization, experimental fish showed an increase in specific antibody levels and lysozyme activities, especially the Fba group. After a lethal challenge with Phdp strain AOD105021, the Fba group achieved the highest relative percentage of survival rate (70.21%) and a significantly lower bacterial load in the spleens than other groups 3 days after infection. The results suggest that Fba is a good candidate for subunit vaccine development against photobacteriosis in fish.

Topics: Animals; Antibodies, Bacterial; Aquaculture; Bacterial Load; Bacterial Proteins; Bacterial Vaccines; Fish Diseases; Fructose-Bisphosphate Aldolase; Gram-Negative Bacterial Infections; Muramidase; Perciformes; Photobacterium; Recombinant Proteins; Spleen; Vaccination; Vaccine Efficacy; Vaccines, Subunit

Aloe vera is a traditional medicinal plant; however, its use in fish is fairly recent. We evaluated the effects of dietary A. vera on stress, innate immunity, and energy metabolism in pacu inoculated with Aeromonas hydrophila. For 7 days, 192 fish were fed with diets supplemented with 0% (control), 0.5%, 1.0%, and 2.0% of the plant extract and then inoculated with bacteria and sampled 3, 6, and 24 h later. All concentrations of A. vera reduced basal levels of cortisol, and 1.0% reduced cortisol levels more intensely 3 h after inoculation. A. vera increased the basal respiratory activity of leukocytes/RAL (0.5 and 1.0%), increased the serum levels of lysozyme (1.0 and 2.0%) 6 h after inoculation, and increased the activity of the complement system after 3 h. Spleen somatic index/SSI increased with 1.0 and 2.0% A. vera. A. vera also promoted metabolic effects. It increased basal levels of lipids in the liver and muscle, as well as hepatosomatic index (1.0%) and, 3 h after inoculation, prevented the reduction of serum triglyceride (1.0%) and reduced the mesenteric fat (1.0%). Bacterial inoculation increased RAL from 3 to 24 h and lysozyme levels at 24 h, increased serum cholesterol at 24 h, and decreased serum triglyceride from 3 to 24 h, regardless of A. vera. We concluded that A. vera offered for only 7 days had stress-reducing effects, stimulated innate immunity, protected triglyceride levels in blood, lipid depots in the liver and muscle, and directed the energy mobilization to visceral depots.

Topics: Aeromonas hydrophila; Aloe; Animals; Blood Glucose; Characiformes; Cholesterol; Energy Metabolism; Fish Diseases; Glycogen; Gram-Negative Bacterial Infections; Hydrocortisone; Immunity, Innate; Leukocytes; Lipids; Liver; Muramidase; Muscles; Plant Extracts; Random Allocation; Reactive Oxygen Species; Stress, Physiological; Triglycerides

This study evaluated the effectiveness of dietary Ziziphus mauritiana leaf powder (ZLP) to control Aeromonas hydrophila infection in Nile tilapia and reduce damage to vital immune organs. Four experimental groups were fed a diet supplemented with ZLP at concentrations of 0, 5, 10, and 20 g/kg (w/w) for 6 weeks. At the end of the feeding trial, all groups were intraperitoneally injected with pathogenic A. hydrophila. It was found that Z. mauritiana significantly (P < 0.05) upregulated (lysozyme, interleukin 1 beta) and superoxide dismutase gene expressions as well as improved the activity of serum lysozyme and liver antioxidant enzymes. The fish that were fed a ZLP-supplemented diet also exhibited significantly higher survival rates after A. hydrophila challenge than those that were fed a ZLP-free diet (P < 0.05). Supplementation of 10 g/kg ZLP most effectively reduced the histopathological alterations caused by A. hydrophila challenge in the liver, spleen, kidney, and muscle of the fish. In conclusion, ZLP can be effective in controlling A. hydrophila infection in Nile tilapia (particularly at a concentration of 10 g/kg) through enhancement of its immune and antioxidant status.

Topics: Aeromonas hydrophila; Analysis of Variance; Animals; Aquaculture; Cichlids; Dietary Supplements; Fish Diseases; Gram-Negative Bacterial Infections; Interleukin-1beta; Kidney; Liver; Muramidase; Muscles; Plant Extracts; Plant Leaves; Random Allocation; Spleen; Superoxide Dismutase; Up-Regulation; Ziziphus

Topics: Aeromonas hydrophila; Animals; Arguloida; Carps; Cloning, Molecular; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Immunity, Innate; Immunization; Liver; Muramidase; Phylogeny; Poly I-C; RNA, Double-Stranded

Over the last 50 years, various approaches have been established for the development of antigens for immunostimulation. We used phage lysate (PL), composed of inactivated antigens by the lytic bacteriophage pAh 6-c for Aeromonas hydrophila JUNAH strain to develop a vaccine for the prevention of A. hydrophila infection in Cyprinus carpio (common carp). We also assessed the poly

Topics: Aeromonas hydrophila; Agglutination Tests; Animals; Antibodies, Bacterial; Bacteriophages; Carps; Gram-Negative Bacterial Infections; Immunization; Interleukin-1beta; Muramidase; Tumor Necrosis Factor-alpha; Vaccination

Topics: Animal Feed; Animals; Bass; Blood Bactericidal Activity; Blood Cell Count; Body Weight; Complement Pathway, Alternative; Disease Resistance; Dose-Response Relationship, Drug; Erythrocyte Indices; Fish Diseases; Gene Expression Profiling; Gram-Negative Bacterial Infections; Hemoglobins; Hydrocortisone; Immunity, Humoral; Inflammation; Muramidase; Neuroimmunomodulation; Nutritional Requirements; Peroxidases; Photobacterium; Tryptophan

Dabry's sturgeon (Acipenser dabryanus) is mainly distributed in the upper Yangtze River. Although extensively farmed, little information is available on its innate immune system. In this study, we conducted de novo transcriptome assembly of the head kidney to create a comprehensive dataset for A. dabryanus. A total of 51,324,686 high quality reads were obtained from head kidney cDNA library by the Illumina sequencing platform and 131,261 unigenes were determined to contain complete ORFs. The complete coding sequences of g- and c-type lysozymes were identified from unigenes, and designated as ADLysG and ADLysC. Aeromonas hydrophila infection of Dabry's sturgeon caused a significant increase (P < 0.05) in blood for both lysozyme types, confirming their active defensive role against bacterial infections. This research provides the first characterization of these enzymes in an ancestral chondrostean. These data suggest that ADLysG and ADLysC have the potential for immune defense system against bacterial infection.

Topics: Aeromonas hydrophila; Amino Acid Sequence; Animals; Fish Diseases; Fish Proteins; Fishes; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Sequence Alignment

The present study was designed to assess the effect of sublethal concentrations of electoplating industry effluent (EIE) on the non-specific and specific immune responses in the freshwater fish, Cyprinus carpio. Sublethal concentrations of electroplating industry effluent such as 0.004, 0.007, 0.010 and 0.013% were chosen based on the LC

Topics: Aeromonas hydrophila; Agglutination Tests; Animals; Antibody Formation; Carps; Electroplating; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Immunity, Innate; Industrial Waste; Muramidase; Peroxidase; Protease Inhibitors; Water Pollution, Chemical

The dietary supplementation of synbiotic in Cirrhinus mrigala juvenile (with initial body weight ranging from 2.87 ± 0.01 g to 3.26 ± 0.05 g) was evaluated in terms of changes in innate immunity, antioxidant activity and disease resistance against Aeromonas hydrophilla infection. One hundred eighty acclimatized juveniles of mrigal were randomly distributed in the three replicates of each of four experimental groups i.e. control (without Probiotic and Prebiotic), T

Topics: Aeromonas hydrophila; Animals; Bacillus subtilis; Cyprinidae; Disease Resistance; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Probiotics; Synbiotics

In the present study, effect of dietary Withania sominefera (W. sominefera) root powder was evaluated to modulate immune and antioxidant response against Aeromonas hydrophila (A. hydrophila) infection in Nile tilapia (Oreochromis niloticus). W. sominefera root powder supplemented diets at two concentrations 2.5% (W 2.5%) and 5% (W 5%); fed for 6 weeks prior to the A. hydrophila challenge and continued the same respective diets during the post challenge period (2 weeks). Results showed that fish fed W. sominefera at 5% enhanced immune response in both pre and post-challenge period. NBT level exhibited only significant increase (P < 0.05) in the pre-challenge period compared to control. Malondialdehyde (MDA) levels in liver and muscle revealed significant decrease in both Withania supplemented groups compared to the control in post challenge period. Antioxidant enzymes activities (catalase/CAT/, glutathione S-transferase/GST/, glutathione/GSH; and superoxide dismutase/SOD) were improved in liver and muscle in post challenge period. Glutathione peroxidase (GPx) level in muscle and serum total antioxidant capacity (TAC) showed a significant increase in both Withania supplemented groups compared to the control post challenge. Withania supplementation enhanced disease resistance against A. hydrophila and reduced mortalities (20%), especially at supplemented concentration of 5%. Our findings suggest that W. sominefera root powder may have protective and immunotherapeutic roles in Nile tilapia against A. hydrophila infection which may be useful in controlling important fish bacterial diseases.

Topics: Aeromonas hydrophila; Animals; Blood Cells; Catalase; Cichlids; Diet; Fish Diseases; Glutathione; Glutathione Peroxidase; Glutathione Transferase; Gram-Negative Bacterial Infections; Liver; Malondialdehyde; Muramidase; Muscles; Nitric Oxide; Phagocytosis; Plant Roots; Protective Agents; Respiratory Burst; Superoxide Dismutase; Withania

The present study investigated the effects of dietary supplementation of yeast nucleotides on the hematology, antioxidant activity, non-specific immunity, expression of intestinal cytokines, and disease resistance in Nile Tilapia (Oreochromis niloticus). Fish weighing 42.90 ± 0.14 g were randomly divided into four groups. Each group was set in triplicate (15 fish per replicate). Fish were fed on four dietary levels of yeast nucleotides (NTs) supplemented with the basal diet 0% (control), 0.05%, 0.15%, and 0.25% NTs. Significantly higher total serum protein, albumin, total serum globulin, total WBC counts, and lymphocyte and granulocyte contents were recorded in 0.25% NT group as compared to the control. The albumin/globulin ratio (A:G) showed a considerable decrease in the 0.25% NT group. The non-specific immune parameters; serum killing percentage, lysozyme activity, nitric oxide assay, IgM levels, and anti-protease activity, were significantly higher in the 0.25% NT group as compared to the control. Moreover, a 15-day feeding trial demonstrated improved results in terms of serum lysozyme activity, nitric oxide assay, IgM levels, and anti-protease activity than a 30-day feeding trial. A significant increase in the anti-oxidant status of O. niloticus was noticed, as reflected by increased superoxide dismutase and malondialdehyde activity in the serum of 0.25% NT group compared to the control, while glutathione peroxidase displayed a significant increase in all groups as compared to the control. The intestinal cytokines TGF-β, IL-1β, IL-10β, and TNF-α mRNA levels showed a pattern of 0.25% NT > 0.15% NT > 0.05% NT > 0% NT, as relative to the control Ef-1α levels. The relative survival percentages of fish fed on yeast nucleotide-supplemented diets, as analyzed by exposure to Aeromonas sobria, were significantly better than the control group. In conclusion, dietary yeast nucleotide administration at 0.25% improved blood proteins, leukocytes, antioxidant activity, non-specific immunity, cytokine gene expression, and disease resistance of Nile Tilapia.

Topics: Aeromonas; Animals; Cichlids; Cytokines; Diet; Disease Resistance; Fish Diseases; Gene Expression; Gram-Negative Bacterial Infections; Immunoglobulin M; Intestines; Leukocyte Count; Malondialdehyde; Muramidase; Nitric Oxide; Nucleotides; RNA, Messenger; Saccharomyces cerevisiae; Superoxide Dismutase

The outer-membrane proteins (OMPs) of Aeromonas hydrophila, an imperative fish pathogen accountable for massive economic losses to aquaculture industry, are found to be immunogenic and considered as potential vaccine candidates. In spite of development in the formulation of vaccine candidates against Aeromonas infection, no commercial preparation has been done so far; in addition, the molecular mechanisms of immunoprotection induced by various vaccine formulations in Indian major carp, Labeo rohita, are little known. The present study was undertaken to evaluate the modulation of immunity and expression of immune-related genes post-rOmpF (recombinant outer-membrane protein of A. hydrophila, a novel vaccine candidate) immunization and protective efficacy after A. hydrophila challenge. The rOmpF-immunized fish showed a variable expression of the immune-related genes, viz. toll-like receptor 22 (TLR), complement component 3 (C3), chemokine (CXCa), tumor necrosis factor-α (TNFα), interleukin 1β (IL-1β), manganese superoxide dismutase (MnSOD) and natural killer enhancing factor (NKEF) in the head kidney tissues, when compared to the control group at different time intervals post-vaccination. A significant increase in serum hemolysin titer, ceruloplasmin level and myeloperoxidase activity was observed on day 140 post immunization. Also, bacterial agglutination titer and antiprotease activity were significantly increased on day 42 post immunization. No significant change was observed in lysozyme activity. Challenge studies with live A. hydrophila on day 140 post-immunization of L. rohita significantly increased the relative percentage survival (∼44%) in the vaccinated group. The results suggest that the rOmpF could be used as a potential vaccine candidate to combat A. hydrophila infection in fish.

Topics: Aeromonas hydrophila; Animals; Antibodies, Bacterial; Bacterial Outer Membrane Proteins; Bacterial Vaccines; Ceruloplasmin; Cyprinidae; Fish Diseases; Gram-Negative Bacterial Infections; Hemolysis; Muramidase; Peroxidase; Porins; Recombinant Proteins

Topics: Aeromonas veronii; Animals; Antigens, Bacterial; Bacterial Vaccines; Carps; Fish Diseases; Gram-Negative Bacterial Infections; Immunoglobulin M; Interleukin-1beta; Muramidase; Tumor Necrosis Factor-alpha; Vaccines, Inactivated

It is well known that lysozymes are key proteins to teleosts in the innate immune system and possess high bactericidal properties. In the present study, a g-type lysozyme gene was cloned from Microptenus salmoides. The g-type sequence consisted of 582 bp, which translated into a 193 amino acid (AA) protein (GenBank accession no: MH087462). The predicted molecular weight and theoretical isoelectric point were 21.36 kDa and 6.91 respectively and no signal peptide was observed. The qRT-PCR analysis showed that the g-type lysozyme gene was differentially expressed in various tissues under normal conditions and the highest g-type lysozyme level was observed in liver, gill and spleen while there seemed to be low expression in the muscle, heart and head-kidney. The expression of g-type lysozyme was differentially upregulated in the spleen, gill and intestine after stimulation with heat stress and Aeromonas hydrophila (A. hydrophila). Under heat stress and A. hydrophila injection, the g-type lysozyme mRNA levels all in spleens, gill and intestine tissues increased significantly (P < 0.05), with the maximum levels attained at 12 h, 24 h (or 12 h) and 24 h. Thereafter, they all decreased significantly (P < 0.01) and the expression in gill returned to nearly the basal value within 72 h. Those results suggested that g-type lysozyme was involved in the immune response to heat stress and bacterial challenge. The cloning and expression analysis of the g-type lysozyme provide theoretical basis to further study the mechanism of anti-adverseness in Microptenus salmoides. The g-type lysozyme gene perhaps also played an important role in the immune responses against bacterial invasion.

Topics: Aeromonas hydrophila; Amino Acid Sequence; Animals; Base Sequence; Bass; Fish Diseases; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacterial Infections; Heat-Shock Response; Immunity, Innate; Muramidase; Phylogeny; Sequence Alignment

This study aimed to investigate the impacts of dietary threonine on intestinal immunity and inflammation in juvenile grass carp. Six iso-nitrogenous semi-purified diets containing graded levels of threonine (3·99-21·66 g threonine/kg) were formulated and fed to fishes for 8 weeks, and then challenged with Aeromonas hydrophila for 14 d. Results showed that, compared with optimum threonine supplementation, threonine deficiency (1) decreased the ability of fish against enteritis, intestinal lysozyme activities (except in the distal intestine), acid phosphatase activities, complement 3 (C3) and C4 contents and IgM contents (except in the proximal intestine (PI)), and it down-regulated the transcript abundances of liver-expressed antimicrobial peptide (LEAP)-2A, LEAP-2B, hepcidin, IgZ, IgM and β-defensin1 (except in the PI) (P<0·05); (2) could up-regulate intestinal pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8 and IL-17D mRNA levels partly related to NF-κB signalling; (3) could down-regulate intestinal anti-inflammatory cytokine transforming growth factor (TGF)-β1, TGF-β2, IL-4/13A (not IL-4/13B) and IL-10 mRNA levels partly by target of rapamycin signalling. Finally, on the basis of the specific growth rate, against the enteritis morbidity and IgM contents, the optimum threonine requirements were estimated to be 14·53 g threonine/kg diet (4·48 g threonine/100 g protein), 15.05 g threonine/kg diet (4·64 g threonine/100 g protein) and 15·17 g threonine/kg diet (4·68 g threonine/100 g protein), respectively.

Topics: Aeromonas hydrophila; Animals; Antimicrobial Cationic Peptides; Blood Proteins; Carps; Cytokines; Diet; Down-Regulation; Enteritis; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Hepcidins; Immunoglobulin M; Intestines; Muramidase; NF-kappa B; Signal Transduction; Threonine; TOR Serine-Threonine Kinases; Up-Regulation

Lysozymes as an important immune factor, play vital roles in innate immune response against pathogen infection. In the present study, one c-type and g-type lysozymes were identified from Chinese giant salamander (Andrias davidianus). They shared highly conserved structural features with lysozymes from other species. Spatial expression analysis revealed that AdlysC transcript was most abundant in liver and stomach, and least in muscle and brain. In contrast, the expression level of AdlysG was most abundant in liver and least in muscle and skin. The transcription level of c-type and g-type lysozymes were up-regulated after Aeromonas hydrophila infection in liver and spleen, indicating their participations in the immune response. Moreover, the recombinant AdlysC and AdlysG protein were produced and purified, and were used to investigate the lysozyme activity at different pH and temperatures. The optimal lytic activity was determined at pH 6.0 and at a temperature of 30 °C. Through the minimal inhibitory concentration test, the rAdlysC and rAdlysG exhibited apparent antibacterial activity against both Gram-positive and Gram-negative bacteria with a variable concentration. In conclusion, it is the first report of lysozymes in A. davidianus, and c-type and g-type lysozymes should be involved in the innate immune response of A. davidianus.

Topics: Aeromonas hydrophila; Animals; Anti-Infective Agents; Bacteriolysis; Chickens; Geese; Gram-Negative Bacterial Infections; Hydrogen-Ion Concentration; Immunity, Innate; Liver; Muramidase; Protein Isoforms; Temperature; Transcriptome; Urodela

The immunostimulatory effect of phospholipopeptide biosurfactant from Staphylococcus hominis (GenBank Accession No: KJ564272) was assessed with Oreochromis mossambicus. The non-specific (serum lysozyme activity, serum antiprotease activity, serum peroxidase activity and serum bactericidal activity), specific (bacterial agglutination assay) immune responses and disease resistance activity against Aeromonas hydrophila were examined. Fish were intraperitonially injected with water soluble secondary metabolite (biosurfactant) of S. hominis at a dose of 2 mg, 20 mg and 200 mg kg(-1) body weight. Commercial surfactant surfactin (sigma) at 20 mg kg(-1) was used as standard and saline as negative control. All the doses of water soluble biosurfactant tested, significantly enhanced the specific, nonspecific immunity and disease resistance from the day of post administration of phospholipopeptide biosurfactant till the tail of the experimental period. These results clearly indicated that the secondary metabolite isolated from S. hominis stimulates the immunity of finfish thereby could enhance aquaculture production.

Topics: Aeromonas hydrophila; Agglutination Tests; Animals; Aquaculture; Disease Resistance; Fish Diseases; Gram-Negative Bacterial Infections; Immunization; Lipoproteins; Muramidase; Peptides; Peroxidase; Protease Inhibitors; Staphylococcus hominis; Surface-Active Agents; Tilapia

Lysozyme as an important nonspecific immune factor, can kill bacteria by hydrolyzing β-1,4-glycosidic linkages of peptidoglycan layer, and plays an important role in innate immune response against pathogen infection. In the present study, we report molecular cloning, tissue distribution and functional characterization of the c-type and g-type lysozymes in Qihe crucian carp Carassius auratus (designated as Ca-clys and Ca-glys, respectively). The full-length of Ca-clys and Ca-glys cDNA were cloned using RT-PCR and RACE methods. Catalytic and other conserved residues, required for functionality, were identified by multiple sequence alignment and structure predicted. The findings indicating the Ca-clys with signal peptide sequence, while the Ca-glys without, imply that the two isozymes function in different sites of cell. Phylogenetic analysis revealed that Ca-clys and Ca-glys genes evolve at different rates. Moreover, spatial expression analysis showed that Ca-clys transcript was most abundant in kidney and least in gill. However, the expression level of Ca-glys was significantly lower compared with Ca-clys in various tissues, which was the most abundant in spleen and least in brain. After intraperitoneal injection with A. hydrophila and lipopolysaccharide (LPS), the mRNA levels of Ca-clys and Ca-glys were generally up-regulated in liver and gill, but indicated the different expression changes in spleen, kidney and head kidney. With regard to the lysozyme activity, it was showed that the total enzyme activities generally increased in liver, gill, spleen, and head kidney after stimulation. These results confirmed that both Ca-clys and Ca-glys play an important role in non-specific immunity after A. hydrophila invasion. In this study, it was speculated that expressions of Ca-clys and Ca-glys were regulated in different patterns against pathogens.

Topics: Aeromonas hydrophila; Amino Acid Sequence; Animals; Base Sequence; Fish Diseases; Fish Proteins; Goldfish; Gram-Negative Bacterial Infections; Immunity, Innate; Lipopolysaccharides; Molecular Conformation; Muramidase; Phylogeny; Sequence Alignment; Transcriptome

The aim of this study was to investigate the protective effects of dietary administration of commercial prebiotic, Immunogen, on immunological indicators, enzymatic responses and stress tolerance in juvenile (81.65 ± 1.49) rainbow trout (Oncorhynchus mykiss) following Aeromonas hydrophila infection. The first group of fish was fed with the diet containing 2 g kg(-1) Immunogen whilst the control group received the diet free of Immunogen. There were three replicates per group. After 6 weeks feeding, the control group were divided into two treatments injected with saline buffer (control), and 1.5 × 10(8) CFU A. hydrophila respectively. The fish fed with the Immunogen supplemented diet were also injected with 1.5 × 10(8) CFU A. hydrophila. Our results revealed that dietary Immunogen increased the level of white blood cell (WBC) and percentage of lymphocyte (P < 0.05), however, the level of red blood cell (RBC), Hematocrit (Hct), hemoglobin (Hb) and percent of monocyte decreased in Untreated-Challenged group but unaffected in the group fed with Immunogen (P < 0.05). The level of lysozyme, alternative complement, antiprotease activity, total protein, albumin and globulin decreased in Untreated- Challenged group compared to control group. However, there was an increase in the level of lysozyme, alternative complement, antiprotease activity, bactericidal activity, in the Treated- Challenged group compared to other groups (P < 0.05). Serum alkali phosphatase (ALT) and aspartate aminotransferase, significantly increased fallowing challenge with A. hydrophila but in the Treated-Challenged group, there was no significant difference compared to the control group (P < 0.05). Lactate dehydrogenase (LDH) level was not different between groups (P > 0.05). Serum cortisol and glucose levels were higher in the challenge group, but these levels were lower in fish under challenge that were fed Immunogen-supplemented diet in contrast to the group fed control diet. The stress responses affected by A. hydrophila challenge (P < 0.05). Serum sodium, potassium and calcium concentration decreased by A. hydrophila exposure (P < 0.05), and Immunogen showed protection effect against this change.

Topics: Aeromonas hydrophila; Animal Feed; Animals; Diet; Disease Resistance; Fish Diseases; Fish Proteins; Gene Expression Regulation; Gram-Negative Bacterial Infections; Head Kidney; HSP70 Heat-Shock Proteins; Immunity, Humoral; Muramidase; Oncorhynchus mykiss; Prebiotics; Tumor Necrosis Factor-alpha

Recent microbiological data have revealed that Gram-negative bacteria are able to protect themselves against the lytic action of host lysozymes by secreting proteinaceous inhibitors. Four distinct classes of such inhibitors have been discovered that specifically act against c-type, g-type and i-type lysozymes. Here, the 1.24 Å resolution crystal structure of the periplasmic i-type lysozyme inhibitor from Aeromonas hydrophila (PliI-Ah) in complex with the i-type lysozyme from Meretrix lusoria is reported. The structure is the first to explain the inhibitory mechanism of the PliI family at the atomic level. A distinct `ridge' formed by three exposed PliI loops inserts into the substrate-binding groove of the lysozyme, resulting in a complementary `key-lock' interface. The interface is principally stabilized by the interactions made by the PliI-Ah residues Ser104 and Tyr107 belonging to the conserved SGxY motif, as well as by the other conserved residues Ser46 and Asp76. The functional importance of these residues is confirmed by inhibition assays with the corresponding point mutants of PliI-Ah. The accumulated structural data on lysozyme-inhibitor complexes from several classes indicate that in all cases an extensive interface of either a single or a double `key-lock' type is formed, resulting in highly efficient inhibition. These data provide a basis for the rational development of a new class of antibacterial drugs.

Topics: Aeromonas hydrophila; Amino Acid Sequence; Animals; Bacterial Proteins; Bivalvia; Crystallography, X-Ray; Gram-Negative Bacterial Infections; Models, Molecular; Molecular Sequence Data; Muramidase; Protein Conformation; Sequence Alignment

This study was conducted to test the hypothesis that elevated dietary arginine enhances immunity of juvenile Jian carp (Cyprinus carpio var. Jian). Fish were fed graded levels of dietary arginine for 9 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila. Head kidney and spleen weights, as well as erythrocyte and leukocyte counts were significantly influenced by dietary arginine levels. A similar trend was also observed for hemagglutination titre, serum lysozyme activity, IgM concentration, C3 and C4 content. The highest survival rates following A. hydrophila infection were obtained in fish fed the diets containing arginine at 16.1-21.9 g/kg diet. Phagocytic activity of leukocytes was significantly enhanced by dietary arginine supplementation. In contrast, acid phosphatase activity significantly decreased with dietary arginine levels. Dietary arginine levels did not have a significant effect on the total iron-binding capacity. Gene expression of TNF-α and TGF-β in head kidney significantly increased with dietary arginine levels up to 21.9 g/kg diet, and decreased thereafter. Fish fed the basal diet exhibited the highest IL-10 mRNA expression level. Gene expression of IL-1β and TOR increased with dietary arginine addition, reaching a plateau at 18.5 and 21.9 g arginine/kg diet, respectively. In spleen, higher IL-1β and TNF-α gene expressions were obtained in fish fed the diets containing 24.5 g arginine/kg diet than in fish fed the other dietary treatments. TGF-β mRNA expression levels were significantly lower in fish fed the diets containing ≤21.9 g arginine/kg diet. IL-10 and TOR mRNA expression levels were lower in fish fed 16.1 g arginine/kg diet, while 4E-BP mRNA expression levels increased with dietary arginine levels up to 12.7 g/kg diet and decreased thereafter. Our results indicate that arginine has beneficial effects on regulating mRNA expression of inflammatory cytokines, as well as TOR and 4E-BP and improving humoral and cellular immunity, therefore enhancing disease resistance of fish.

Topics: Aeromonas hydrophila; Animals; Arginine; Carps; Complement C3; Complement C4; Cytokines; Diet; Disease Resistance; Erythrocyte Count; Fish Diseases; Fish Proteins; Gene Expression; Gram-Negative Bacterial Infections; Head Kidney; Immunoglobulin M; Leukocyte Count; Muramidase; Organ Size; Phagocytosis; RNA, Messenger; Spleen; TOR Serine-Threonine Kinases

Enzyme activities and gene expression of a number of innate immune parameters in the serum, mucus and skin of Atlantic salmon (Salmo salar) were investigated after challenge with a pathogenic strain of Aeromonas salmonicida (A. salmonicida). Fish were injected in the dorsal muscle with either 100 μl bacterium solution, about 3.05 × 10(7) CFU/ml A. salmonicida, or 100 μl 0.9% NaCl (as control group) and tissue samples were collected at days 0, 2, 4 and 6 post-injection. Lysozyme (LSZ) and alkaline phosphatase (AKP) activities in serum, mucus and skin, and LSZ and AKP mRNA expression in skin of the challenged fish were higher than those of the control at most of the experimental time, with significant differences at several time points (P < 0.05), indicating the involvement of LSZ and AKP in the innate immunity of Atlantic salmon to A. salmonicida. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities in mucus and skin, along with the SOD, POD and CAT mRNA expression in skin significantly decreased at day 4 and 6, indicating the decreased antioxidant capacity of the challenged fish. Glutamate pyruvate transaminase (GPT) and glutamic oxalacetic transaminase (GOT) activities in serum, mucus and skin of the challenged group were all higher than those of the control after the injection, and at several time points significant differences were found between the two groups, suggesting organs of fish were impaired after the pathogen infection. The changes of the GPT and GOT activities could be used as potential biomarkers for the impairment of physiological functions caused by the pathogen infection. Identified biomarkers of the immune responses will contribute to the early-warning system of the disease. So this study will not only provide a theoretical basis for vaccine development, but also provide basic data for the establishment of early warning systems for diseases caused by A. salmonicida in Atlantic salmon rearing.

Topics: Aeromonas salmonicida; Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Catalase; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Mucus; Muramidase; Peroxidase; RNA, Messenger; Salmo salar; Skin; Superoxide Dismutase

In the present study, KGM was degraded by H2O2 and HCl to obtain two products with different molecular weights: oxidized konjac glucomannan (OKGM, 4.7 × 10(5) Da) and low-molecular-weight oxidized konjac glucomannan (L-OKGM, 9.2 × 10(3) Da). The effects of the two OKGM products on IL-1β, TNF-α, and TLR22 gene expression, and immune parameters and the resistance to Aeromonas hydrophila of Schizothorax prenanti were determined. The results showed that the lysozyme activity was significantly enhanced by the L-OKGM diets. The SOD activity was significantly increased by both OKGM and L-OKGM diets. The MDA level of fish fed the OKGM and L-OKGM diets was significantly lower than the control group. IL-1β mRNA level in the spleen significantly increased in all L-OKGM fed groups. The 8.0 g kg(-1) L-OKGM diet also significantly up-regulated IL-1β gene expression in the head kidney. In the gut, IL-1β mRNA levels were significantly higher in fish fed with the 8.0 g kg(-1) OKGM and 16.0 g kg(-1) L-OKGM diets. The TNF-α mRNA level of L-OKGM group significantly increased in the spleen, head kidney and gut. High dosing of OKGM significantly up-regulated TNF-α transcription in the head kidney, while only the 8.0 g kg(-1) OKGM group showed significantly higher TNF-α mRNA expression in the mesonephros. Fish fed the L-OKGM diets showed significantly higher expression of TLR22 in the spleen, head kidney and mesonephros. After the injection of A. hydrophila, the 8.0 g kg(-1) L-OKGM group showed a significantly higher survival rate than did the control group. Present study suggests that OKGM and L-OKGM can up-regulate immune-related gene expression and enhance disease resistance in S. prenanti, and L-OKGM exhibits higher immunomodulatory activity.

Topics: Aeromonas hydrophila; Animals; Cyprinidae; Dietary Supplements; Disease Resistance; Fish Diseases; Gene Expression; Gram-Negative Bacterial Infections; Head Kidney; Interleukin-1beta; Intestinal Mucosa; Malondialdehyde; Mannans; Muramidase; RNA, Messenger; Spleen; Superoxide Dismutase; Toll-Like Receptors; Tumor Necrosis Factor-alpha

The present study aimed to investigate the effects of Chlorophytum borivilianum polysaccharide (CBP), as a dietary supplement administered at varying concentrations with feed (basal diet), on various cytokine-related responses in Labeo rohita fingerlings. Immune parameters and immune-related gene expressions were measured at 3rd, 4th, and 5th week after feeding. The results revealed that dietary administration of CBP at 0.2% and 0.4% for 4 weeks significantly upregulated serum lysozyme and phagocytic activity. Complement C3 and respiratory burst activity (RBA) were significantly higher after 4 weeks of CBP feeding. The immune related genes IL-8, IL-1β, TNF-α, and iNOS were downregulated (P < 0.05) in groups with 0.2% and 0.4% CBP supplemented diets at week 4. Expression of anti-inflammatory cytokines (IL-10 and TGF-β) was also downregulated (P < 0.5) after 4 weeks of feeding with 0.2% to 0.8% CBP. However, five weeks of CBP administration had no significant effect on immune gene expression, except TNF-α and IL-8. Fish fed with 0.4% CBP for 4 weeks showed maximum resistance against Aeromonas hydrophila (73.3% survival) compared to control. From these results, we recommend that CBP administration at 0.4% for 4 weeks could effectively improve immune response and disease resistance in L. rohita.

Topics: Aeromonas hydrophila; Animal Feed; Animals; Complement C3; Cyprinidae; Dietary Supplements; Disease Resistance; Fish Diseases; Gene Expression Regulation; Gram-Negative Bacterial Infections; Immunity, Innate; Interleukin-10; Interleukin-1beta; Interleukin-8; Liliaceae; Muramidase; Nitric Oxide Synthase Type II; Phagocytosis; Polysaccharides; Respiratory Burst; Survival Analysis; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

In this study, the effects of oral administration of different levels of Dunaliella salina (a natural β-carotene source) on growth parameters, immunological and hematological indices, as well as skin carotenoids, of Heros severus were investigated. One hundred and eighty H. severus weighing 27 ± 0.5 g were divided randomly into four groups in triplicate (15 fish in each replicate). Groups 1-4 received food supplemented with 0, 50, 100 and 200 mg kg⁻¹ D. salina powder, respectively. After 6 weeks, the growth parameters were compared among the groups. Blood samples were taken from each group, and hematological parameters including red blood cell count (RBC), white blood cell count (WBC), hematocrit (PCV), hemoglobin (Hb) and immunological indices (serum and mucus lysozyme and bactericidal activity, resistance against Aeromonas hydrophila infection) as well as carotenoid content of skin were evaluated. Results showed that some growth indices increased significantly in fish fed with 100 and 200 mg kg⁻¹ D. salina-supplemented food (P < 0.05). Although serum lysozyme activity was increased in fish fed with food supplemented with 100 and 200 mg kg⁻¹ D. salina (P < 0.05), no significant change was observed in serum and mucus bactericidal activity and mucus lysozyme activity among the groups (P > 0.05). Most of the hematological parameters such as WBC, RBC, PCV and Hb significantly increased in D. salina-treated fish compared with controls (P < 0.05). Mortality induced after challenge with A. hydrophila in 200 mg kg⁻¹ D. salina-treated fish was 36.67 %, which significantly decreased compared with control (P < 0.05). Skin carotenoid content in all D. salina treatments was statistically higher than that of control (P < 0.05). Conclusively, D. salina as a food additive can affect positively the growth, immunological and hematological parameters of H. severus.

Topics: Aeromonas hydrophila; Animals; Aquaculture; Carotenoids; Chlorophyta; Cichlids; Diet; Gram-Negative Bacterial Infections; Mucus; Muramidase; Random Allocation; Serum Bactericidal Test; Skin; Up-Regulation

The present study was designed to test the immunostimulatory efficacy of poly-β hydroxybutyrate-hydroxyvalerate (PHB-HV) extracted from Bacillus thuringiensis B.t.A102 on the immune system of Oreochromis mossambicus. Fish were fed with 0%, 1%, 3% or 5% PHB-HV supplemented feed and were bled at regular intervals of 5 days. The specific immune response was measured in terms of antibody response to sheep red blood cells, the nonspecific immune mechanisms were analysed in terms of serum lysozyme activity, total peroxidases activity and antiprotease activity. The overall functional immunity was tested by experimental challenge with live virulent Aeromonas hydrophila. The results revealed that all the doses of PHB-HV supplementation in feed were effective in stimulating both specific and nonspecific immune mechanisms. The bacterial challenge experiment showed that highest dose of 5% PHB-HV supplementation was more effective than 1% and 3% doses. The study concludes that PHB-HV can be used as a potential immunostimulant in finfish aquaculture.

Topics: Aeromonas hydrophila; Animals; Antibody Formation; Bacillus thuringiensis; Fish Diseases; Gram-Negative Bacterial Infections; Hemagglutination Tests; Immunization; Male; Muramidase; Peroxidase; Polyesters; Protease Inhibitors; Spectroscopy, Fourier Transform Infrared; Tilapia

The study isolated three strains of intestinal autochthonous bacteria Aeromonas veronii BA-1, Vibrio lentus BA-2, and Flavobacterium sasangense BA-3 from the intestinal tract of the common carp (Cyprinus carpio). To reveal the effects of these three strains of bacteria on the innate immunity of carp, the lysozyme, complement C3, total serum protein, albumin and globulin levels, respiratory burst activity, phagocytic activity by blood leucocytes and the expression of IL-1b, lysozyme-C, and TNF-α were examined after feeding with seven different diets for up to 28 days. Also the survival of carp against Aeromonas hydrophila was challenged for 14 days. The carp were fed seven different diets: one control, three diets supplemented with 1 × 10(8) cell g(-1) of carp intestinal bacteria BA-1 (Group D-I), BA-2 (Group D-II) and BA-3 (Group D-III), and three diets supplemented with extracellular products FA-1 (Group E-I), FA-2 (Group E-II) and FA-3 (Group E-III) which were corresponding to the strains BA-1, BA-2, and BA-3, respectively, up to 28 days. For groups D-I, D-III, E-I and E-III, the innate immune parameters of carp were significantly increased, the expression of three immune-related genes in blood was significantly up-regulated examined during 7, 14, and 21 days of feeding, and the survival rate was improved. The study indicates that the two isolated intestinal autochthonous bacteria A. veronii BA-1 and F. sasangense BA-3 could positively influence immune response and enhance disease resistance of carp against A. hydrophila infection.

Topics: Aeromonas hydrophila; Animals; Blood Proteins; Carps; Complement C3; Fish Diseases; Gene Expression Profiling; Gram-Negative Bacterial Infections; Immunity, Innate; Intestines; Microbiota; Muramidase; Phagocytosis; Random Allocation; Real-Time Polymerase Chain Reaction; Respiratory Burst; RNA

This study investigates the effects of prebiotic Immunogen on lysozyme, TNFα and HSP70 gene expression in head kidney, humoral innate immune parameters and resistant to Aeromonas hydrophila of rainbow trout. 120 healthy rainbow trout (81.65 ± 1.49 g) were distributed in six fiberglass tanks assigned to two groups fed control or diet supplemented with 2 g kg(-1) Immunogen for 45 days. The results revealed that administration of Immunogen significantly (P < 0.05) up regulated lysozyme and TNFα gene expression. HSP70 gene expression was significantly (P < 0.05) lower in Immunogen fed fish at the end of trial. Humoral innate immune parameters (lysozyme activity, ACH50 and bactericidal activity) were significantly (P < 0.05) increased whether 15 or 45 days after feeding on Immunogen supplemented diet. However, significant (P < 0.05) increase in agglutination antibody titer observed just after 45 days feeding on Immunogen. Rainbow trout fed with 2 g kg(-1) Immunogen showed remarkably higher resistance against A. hydrophila (64.44% survival) compared to the control group (24.44% survival). These results confirm that Immunogen can up regulates immune related genes expression, stimulates immune response that per se enhances disease resistance in rainbow trout.

Topics: Aeromonas hydrophila; Animal Feed; Animals; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Fish Proteins; Gene Expression Regulation; Gram-Negative Bacterial Infections; Hematologic Tests; HSP70 Heat-Shock Proteins; Immunity, Innate; Muramidase; Oncorhynchus mykiss; Prebiotics; Real-Time Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

Evaluation of functional mechanism of immunostimulant in fish is essential for realizing their therapeutic potential. The impact of dietary supplementation of seeds of Achyranthes aspera on the immune system of Catla catla was studied. Catla (37.73 ± 6.7 g) were fed with test diets containing 0.5 (D1) and 1.0% (D2) seeds of A. aspera and control diet (D3) for 50 days. Then fish were immunized with heat-killed Aeromonas hydrophila. Blood and tissue samples were collected after 7 days of immunization. Three fish were sacrificed for each parameter. Respiratory burst activity, bactericidal activity and antibody response were significantly (P < 0.05) higher in test diets fed catla compared to the control one. Nitric oxide synthase and serum lysozyme were significantly (P < 0.05) higher in fish fed with diet D2 compared to the other treatments. There were up-regulations of TNFα, lysozyme c and g gene expressions in kidney and IL-10, lysozyme c and g expressions in hepatopancreas of test diets fed fish compared to the control one. The expression of IL-10 was down-regulated in the kidney of D2 diet fed fish compared to others. In the gill, significantly (P < 0.05) higher expressions of TNFα was found in D1 diet feed fish and lysozyme c and in D2 diet fed fish compared to the other groups. This study showed tissue-specific gene expression pattern in catla. An inverse relationship was found between the expression of TNFα and IL-10 in kidney of catla.

Topics: Achyranthes; Aeromonas hydrophila; Animals; Aquaculture; Carps; Fish Diseases; Gene Expression Regulation; Gills; Gram-Negative Bacterial Infections; Hepatopancreas; Immunomodulation; Interleukin-10; Kidney; Muramidase; Nitric Oxide Synthase; Random Allocation; Respiratory Burst; Tumor Necrosis Factor-alpha

The effects of a Chinese herbal mixture (CHM) composed of astragalus, angelica, hawthorn, Licorice root and honeysuckle on immune responses and disease resistant of Nile tilapia (Oreochromis niloticus GIFT strain) were investigated in present study. Fish were fed diets containing 0 (control), 0.5%, 1.0%, 1.5% or 2.0% CHM (w/w) for 4 weeks. And series of immune parameters including lysozyme, cytokine genes TNF-α and IL-1β, superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA) were measured during test period. After four weeks of feeding, fish were infected with Aeromonas hydrophila and mortalities were recorded. Results of this study showed that feeding Nile tilapia with CHM-supplementation diet stimulated lysozyme activity, SOD activity and POD activity in serum, induced TNF-α and IL-1β mRNA expression in head kidney and spleen, but decreased serum MDA content. All CHM-supplemental groups showed reduced mortalities following A. hydrophila infection compared with the group fed the control diet. These results suggested that this CHM can be applied as a tilapia feed supplement to elevate fish immunity and disease resistance against A. hydrophila.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Analysis of Variance; Animal Feed; Animals; Cichlids; Dietary Supplements; DNA Primers; Drugs, Chinese Herbal; Fish Diseases; Gram-Negative Bacterial Infections; Malondialdehyde; Muramidase; Peroxidase; Real-Time Polymerase Chain Reaction; Superoxide Dismutase

Despite the importance and success of developing a candidate vaccine against Aeromonas hydrophila infection in fish, little is known about the molecular mechanisms of the vaccine-induced immunoprotection in Indian major carp, Labeo rohita, primarily due to lack of information on most of the immune related genes of the species. In this study, a novel candidate antigen recombinant outer membrane protein R (rOmpR) of A. hydrophila was evaluated as a vaccine candidate along with a modified adjuvant formulation. Protective efficacy of the rOmpR immunization was assessed in terms of survival against A. hydrophila challenge as well as modulation of immune response in vaccinated fish after 1, 3, 6, 12, 24, 72 h and 10 days post-injection (using immune gene expression analysis) and 10, 28, 56 and 140 days post-injection (serum immune parameter analysis). The generated immune response was compared with a formalin-killed A. hydrophila antigen preparation using mineral oil only and modified adjuvant alone. We report a variable up-regulation of the immune-related genes viz., lysozyme G, complement factor 4, immunoglobulin M, β2-microglobulin, major histocompatibility complex I and II, and interleukin-1β in anterior kidney and spleen tissues at early time points post-immunization in all the groups, when compared to the control fish. The vaccinated fish also showed an increase in serum natural hemolysin titer, lysozyme and myeloperoxidase activities, and antibody titer irrespective of vaccine formulations as compared to control fish on days 10, 28 and 56. However, the increase in the serum parameters was more pronounced on day 140 in rOmpR-modified adjuvant injected group, indicating the modulatory role of this new vaccine formulation. Upon challenge with live A. hydrophila on days 56 and 140 post-immunization, significantly reduced percent mortality was noted in the group immunized with modified adjuvant based rOmpR vaccine formulation. Taken together, our results suggest that rOmpR along with modified adjuvant could potentially be used as a vaccine formulation to handle A. hydrophila infection on a long-term basis.

Topics: Aeromonas hydrophila; Analysis of Variance; Animals; Bacterial Outer Membrane Proteins; Bacterial Vaccines; Carps; DNA Primers; DNA, Complementary; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Peroxidase

This trial was carried out to investigate the effects of dietary administration of Vitacel(®), a commercial fermentable fiber, on immune related genes (Lysozyme, TNFα and HSP70) expression, innate immune response and resistance of rainbow trout against Aeromonas hydrophila. 120 healthy rainbow trout (81.65 ± 1.49 g) were distributed in six fiberglass tanks assigned to two treatments. The treatments were feeding rainbow trout with diets supplemented with 0 (control) or 10 g kg(-1) Vitacel(®) for 45 days. The results revealed that administration of fermentable fiber significantly (P < 0.05) upregulated lysozyme and TNFα gene expression. HSP70 gene expression was significantly lower in Vitacel(®) fed fish at the end of trial (P < 0.05). Furthermore dietary administrations of Vitacel(®) remarkably elevated rainbow trout innate immune parameters include serum lysozyme, ACH50, bactericidal activity and agglutination antibody titer (P < 0.05). Administration of 10 g kg(-1) Vitacel(®) significantly increased rainbow trout resistance against A. hydrophila (P < 0.05). The results of present study revealed that dietary Vitacel(®) can upregulates immune related genes expression and elevates innate immune response and disease resistance of rainbow trout.

Topics: Aeromonas hydrophila; Agglutination Tests; Animals; Dietary Fiber; Disease Resistance; DNA Primers; Fermentation; Fish Diseases; Gene Expression Regulation; Gram-Negative Bacterial Infections; HSP70 Heat-Shock Proteins; Immunity, Innate; Muramidase; Oncorhynchus mykiss; Real-Time Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

Fucoidan is sulfated polysaccharide extracted from seaweed brown algae. This study was designed to evaluate the immunomodulatory effects and disease resistance of dietary fucoidan on catfish, Clarias gariepinus, immunosuppressed by cadmium. Three hundred and sixty African catfish, C. gariepinus, was allocated into six equal groups. The first group served as a control. Groups (F1 and F2) were fed on fucoidan supplemented ration at concentrations of 4 and 6g/kg diet respectively for 21 days. Groups (Cd, CdF1 and CdF2) were subjected throughout the experiment to a sub-lethal concentration of 5ppm cadmium chloride solution and groups (CdF1 and CdF2) were fed on a ration supplemented with fucoidan. Macrophages oxidative burst, phagocytic activity percentages and lymphocytes transformation index were a significant increase in the fucoidan-treated groups (F1 and F2), while serum lysozyme, nitric oxide and bactericidal activity were enhanced only in group (F2) when compared with controls. These parameters as well as absolute lymphocyte count and survival rate were significantly increased in group (CdF2) when compared with cadmium chloride immunosuppressed group (Cd). It could be concluded that the fucoidan can be used as immunostimulant for the farmed African catfish, C. gariepinus as it can improve its resistance to immunosuppressive stressful conditions.

Topics: Aeromonas hydrophila; Animals; Aquaculture; Cadmium Chloride; Catfishes; Dietary Supplements; Fish Diseases; Gram-Negative Bacterial Infections; Immunocompromised Host; Immunosuppressive Agents; Kaplan-Meier Estimate; Leukocyte Count; Muramidase; Nitric Oxide; Phagocytosis; Polysaccharides; Random Allocation; Respiratory Burst

To understand whether chicken-type lysozyme (Lys-c) in channel catfish was induced by infection of Aeromonas hydrophila, the transcriptional levels of Lys-c in skin, gut, liver, spleen, posterior kidney, and blood cells in healthy channel catfish was compared to that in channel catfish infected with A. hydrophila by bath immersion. Quantitative PCR revealed that the transcription levels of Lys-c in infected catfish were significantly (P < 0.05) induced in all five tissues tested as well as in blood cells. Recombinant CC-Lys-c produced in Escherichia coli expression system (R-CC-Lys-c) exhibited significant (P < 0.05) lytic activity to Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-c (pcDNA-Lys-c) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-c offered significant (P < 0.05) protection to G1B against A. hydrophila infection. When channel catfish were intraperitoneally injected with QCDCR adjuvant formulated pcDNA-Lys-c and challenged with a highly virulent A. hydrophila strain AL-09-71 at 1-, 2-, 14-, and 28-days post treatment, pcDNA-Lys-c offered 75%, 100%, 60%, and 77% protection to channel catfish, respectively. Macrophages of fish treated with pcDNA-Lys-c produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish treated with pcDNA vector alone. Taken together, our results suggest that pcDNA-Lys-c could be used as a novel immunostimulant to protect channel catfish against A. hydrophila infection.

Topics: Aeromonas hydrophila; Animals; Fish Diseases; Fish Proteins; Gene Expression Regulation; Gram-Negative Bacterial Infections; Ictaluridae; Muramidase; Nitric Oxide; Phylogeny; Reactive Oxygen Species

The present study evaluated the effect of dietary andrographolide (EC 50%) on growth, non-specific immune parameters and disease resistance against Aeromonas hydrophila infection in Indian major carp, Labeo rohita fingerlings. Fishes were fed with formulated diet containing andrographolide as T0 (0.00%), T1 (0.05%), T2 (0.10%), T3 (0.20%), T4 (0.40%) and T5 (0.80%) for 42 days. Fishes were challenged with A. hydrophila 42 days post feeding and relative percentage survival (RPS) was recorded over 14 days post challenge. Blood and serum samples were collected for nonspecific immune parameters on 14, 28 and 42 days of feeding and growth performance was evaluated at the end of experiment. The results revealed that fishes fed with andrographolide showed significant (p < 0.05) increase in NBT levels, myeloperoxidase activity, phagocytic activity, serum lysozyme activity, and serum antiprotease activity when compared to the control group. The weight gain, specific growth rate, feed conversion ratio and protein efficiency ratio of fishes fed with andrographolide were found to be significantly (p < 0.05) differed compared with control. Dietary andrographolide at the level of 0.10% showed significantly (P < 0.05) higher RPS (74.06%) against A. hydrophila infection than control. The results revealed that andrographolide supplemented diet has a stimulatory effect on non-specific immune parameters along with improved growth performance and increased disease resistance against A. hydrophila infection in L. rohita fingerlings.

Topics: Aeromonas hydrophila; Analysis of Variance; Animals; Aquaculture; Cyprinidae; Dietary Supplements; Disease Resistance; Diterpenes; Dose-Response Relationship, Drug; Fish Diseases; Gram-Negative Bacterial Infections; Muramidase; Nitroblue Tetrazolium; Peroxidase; Phagocytosis; Protease Inhibitors

The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme-g (CC-Lys-g) produced in Escherichia coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme-g plasmid DNA could be used as an immunostimulant to protect channel catfish against Aeromonas hydrophila infection. Recombinant CC-Lys-g produced in E. coli expression system exhibited significant (P < 0.05) lytic activity against Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-g (pcDNA-Lys-g) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-g offered significant (P < 0.05) protection to G1B cells against A. hydrophila infection. When channel catfish were intraperitoneally injected with pcDNA-Lys-g along with an adjuvant QCDCR, the transcriptional level of Lys-g was significantly (P < 0.05) increased. When pcDNA-Lys-g injected fish was challenged with a highly virulent A. hydrophila strain AL-09-71, pcDNA-Lys-g offered 100% protection to channel catfish at two days post DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish injected with pcDNA vector alone at two days post DNA injection. Taken together, our results suggest that pcDNA-Lys-g could be used as a novel immunostimulant to offer immediate protection to channel catfish against A. hydrophila infection.

Topics: Actinomycetales Infections; Adjuvants, Immunologic; Aeromonas hydrophila; Amino Acid Sequence; Animals; Cloning, Molecular; DNA, Complementary; Escherichia coli; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Ictaluridae; Micrococcus; Molecular Sequence Data; Muramidase; Phylogeny; Plasmids; Real-Time Polymerase Chain Reaction; Recombinant Proteins; RNA, Messenger; Sequence Alignment

To determine whether transcriptional levels of channel catfish (Ictalurus punctatus) genes are differentially regulated between a first infection with Aeromonas hydrophila and a re-infection, suppression subtractive hybridization (SSH) was performed in this study using anterior kidney cDNA after the re-infection as tester. Of the 96 clones isolated from the SSH library, 28 unique expressed sequence tags (ESTs) were obtained, of which eight were confirmed to be slightly but significantly (P < 0.05) more up-regulated by the re-infection at 6 h post infection (hpi). Expression kinetics studies at 3, 6, 12, 24, and 48 hpi revealed that the eight ESTs were significantly (P = 0.016) more up-regulated by the first infection, with a major peak at 3 hpi. A total of 96 genes reported in literature to be up-regulated by bacterial infections were selected and subjected to expression analysis at 3 hpi. Of the 96 selected genes, 19 were found to be significantly (P < 0.05) induced by A. hydrophila after the first infection and the re-infection. The 19 genes belonged to the following five main categories: 1) toll-like receptor (TLR2, TLR3, TLR5, TLR21); 2) antimicrobial peptide (NK-lysin type 1, NK-lysin type 2, NK-lysin type 3, cathepsin D, transferrin, hepcidin); 3) cytokine or chemokine (interleukin-1β, interleukin-10, tumor necrosis factor α, chemokine CXCL-10); 4) signaling proteins (cadherin EGF LAG seven-pass G-type receptor 1, very large inducible GTPase 1, arginine deiminase type 2, lymphokine-activated killer T-cell originated protein kinase); 5) lysozyme (lysozyme c). Overall, the total 27 genes (8 ESTs plus the 19 selected genes) were significantly (P < 0.001) more induced by the first infection. Peaked expression of lysozyme c and serum lysozyme activity after the first infection were seen at 24 hpi, whereas that after the re-infection were seen at 12 hpi, suggesting that both innate and adaptive immunity were involved in the defense against the re-infection of A. hydrophila.

Topics: Aeromonas hydrophila; Animals; Antimicrobial Cationic Peptides; Base Sequence; Cytokines; DNA Primers; Expressed Sequence Tags; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Gene Library; Gram-Negative Bacterial Infections; Ictaluridae; Molecular Sequence Data; Muramidase; Sequence Analysis, DNA; Statistics, Nonparametric; Toll-Like Receptors

A bacterial strain isolated from intestines of hybrid catfish (Clarias gariepinus Male × Clarias macrocephalus Female) exhibited an in vitro inhibitory effect on a fish pathogen, Aeromonas hydrophila TISTR 1321. By using the 16S rDNA sequence analysis, it was identified as Lactobacillus plantarum C014. To examine whether L. plantarum C014 had potential for use as an immunostimulant and biocontrol agent in hybrid catfish, the fish diet supplemented with L. plantarum C014 (10(7) CFU/g diet) was prepared and used for the in vivo investigation of its effect on innate immune response and disease resistance of hybrid catfish. Two innate immune response parameters, phagocytic activity of blood leukocytes and plasma lysozyme activity, were significantly enhanced in the treated fish after 45 days of feeding. Feeding the fish with the L. plantarum C014 supplemented diet for 45 days before challenging them with A. hydrophila at the dose of LD50 could reduce the mortality rate of the fish from 50% (in control group) to 0% (in treated group). Based on its origin and beneficial effect on innate immune response and disease resistance, L. plantarum C014 may be a potential candidate for use as a natural and safe immunostimulant and biocontrol agent in hybrid catfish.

Topics: Aeromonas hydrophila; Analysis of Variance; Animals; Base Sequence; Catfishes; Dietary Supplements; Disease Resistance; DNA Primers; Fish Diseases; Gram-Negative Bacterial Infections; Hybridization, Genetic; Immunity, Innate; Lactobacillus plantarum; Lethal Dose 50; Molecular Sequence Data; Muramidase; Phagocytosis; Probiotics; RNA, Ribosomal, 16S; Sequence Analysis, DNA

Lysozyme is a key component of the innate immune system and plays an important role in antibacterial infection. In this study, we analyzed the expression and activity of a chicken-type (c-type) lysozyme (named SmLysC) from turbot (Scophthalmus maximus). SmLysC is composed of 143 residues and shares 67-90% overall sequence identities with the c-type lysozymes of a number of teleost fish. SmLysC possesses a typical c-type lysozyme domain, which contains the conserved residues E50 and D67 that form the putative catalytic site. SmLysC expression was detected, in increasing order, in head kidney, gill, heart, muscle, brain, spleen, blood, and liver. Bacterial infection caused significant inductions of SmLysC expression in head kidney, spleen, and liver in a time-dependent manner. Immunoblot analysis indicated that SmLysC has a subcellular localization in the extracellular milieu. Recombinant SmLysC (rSmLysC) was able to bind to bacterial cells and inhibit bacterial growth. Enzyme assay showed that the optimal temperature and pH of rSmLysC were 37 °C and pH 6.0 respectively. In contrast to rSmLysC, the mutant protein rSmLysCM1, which bears alanine substitutions at E50 and D67, displayed drastically reduced bacteriolytic activity. rSmLysC was able to inhibit the growth of several fish bacterial pathogens in a manner that depended on the dose of the protein; however, Gram-positive bacteria were in general more sensitive to rSmLysC than Gram-negative bacteria. Together these results indicate that SmLysC is a functional lysozyme that is likely to participate in innate immune defense against extracellular bacterial pathogens, in particular those of Gram-positive nature.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Fish Proteins; Flatfishes; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Gram-Positive Bacteria; Gram-Positive Bacterial Infections; Immunoblotting; Muramidase; Organ Specificity; Plasmids; Polymerase Chain Reaction; Recombinant Proteins; Sequence Alignment; Sequence Analysis, DNA; Species Specificity

This study evaluated the effects of dietary cholesterol on antioxidant capacity, non-specific immune response and resistance to Aeromonas hydrophila in rainbow trout (Oncorhynchus mykiss) fed soybean meal-based diets. Fish were fed diets supplemented with graded cholesterol levels (0 [control], 0.3, 0.6, 0.9, 1.2, and 1.5%) for nine weeks. The fish were then challenged by A. hydrophila and their survival rate recorded for the next week. Dietary cholesterol supplementation generally increased the serum and hepatic superoxide dismutase (SOD), glutathione-peroxidase (GSH-Px), catalase (CAT), and total antioxidant capacity (TAC) activities, but decreased the serum and hepatic malondialdehyde (MDA) contents. Further, the hepatic CAT and serum SOD, CAT, and TAC activities were significantly higher in fish fed diets supplemented with 0.9 or 1.2% cholesterol compared to those fed the control diet, whereas the serum and hepatic MDA contents were significantly lower. The respiratory burst activity, alternative complement activity, and hepatic lysozyme activity increased steadily when the supplemental cholesterol was increased by up to 1.2% and then declined with further addition. The serum lysozyme activity and phagocytic activity increased steadily with increasing dietary supplemental cholesterol level up to 0.9% and then declined with further addition. Dietary cholesterol supplementation generally enhanced the protection against A. hydrophila infection, and fish fed diets supplemented with 0.9 or 1.2% cholesterol exhibited the highest post-challenge survival rate. The results indicated that cholesterol may be under-supplied in rainbow trout fed soybean meal-based diets, and dietary cholesterol supplementation (0.9-1.2%) contributed to improved immune response and disease resistance of rainbow trout against A. hydrophila.

Topics: Aeromonas hydrophila; Animal Feed; Animals; Antioxidants; Aquaculture; Cholesterol; Complement Pathway, Alternative; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Oncorhynchus mykiss; Phagocytosis; Respiratory Burst

The effect of Coriolus versicolor extract supplemented diets on innate immune response and disease resistance in kelp grouper, Epinephelus bruneus against Listonella anguillarum, is reported. Kelp grouper were divided into four groups of 25 each and fed with C. versicolor enriched diets at 0% (control), 0.01%, 0.1%, and 1.0% level. After 30 days of feeding, all fish were injected interaperitoneally (i.p.) with 50 μl of L. anguillarum (4.7 × 10(7) CFU) to investigate the immune parameters at weeks 1, 2, and 4. The reactive oxygen species and reactive nitrogen species production were significantly enhanced in fish fed with 0.1% and 1.0% supplementation diets from weeks 1-4 when compared to the non enriched diet fed and infected control. The phagocytic activity significantly increased with 0.1% and 1.0% diets on weeks 2 and 4. The leucocyte myeloperoxidase content, lysozyme activity, and total protein level significantly increased when fed with 0.1% and 1.0% supplementation diets from weeks 1-4. The cumulative mortality was 35% and 45% in 1.0% and 0.1% enriched diet fed groups whereas it was 55% and 80% in 0.01% and 0% groups respectively. The present results suggest that diets enriched with C. versicolor at 0.1% or 1.0% level positively enhance the innate immune system and affords protection from L. anguillarum.

Topics: Animals; Bass; Blood Proteins; Dietary Supplements; Disease Resistance; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Leukocytes; Listonella; Muramidase; Peroxidase; Phagocytosis; Reactive Nitrogen Species; Reactive Oxygen Species; Time Factors

Lysozyme is an important molecule of innate immune system for the defense against bacterial infections. Three genes encoding chicken-type (c-type) lysozymes, C1-, C2-, C3-type, were obtained from tilapia Oreochromis aureus by RT-PCR and the RACE method. Catalytic and other conserved structure residues required for functionality were identified. The amino acid sequence identities between C1- and C2-type, C1- and C3-type, C2- and C3-type were 67.8%, 65.7% and 63.9%, respectively. Phylogenetic tree analyze indicated the three genes were firstly grouped to those of higher teleosteans, Pleuronectiformes and Tetraodontiformes fishes, and then clustered to those of lower teleosteans, Cypriniformes fishes. Bioinformatic analysis of mature peptide showed that the three genes possess typical sequence characteristics, secondary and tertiary structure of c-type lysozymes. The three tilapia c-type lysozymes mRNAs were mainly expressed in liver and muscle, and C1-type lysozyme also highly expressed in intestine. C1-type lysozyme mRNA was weakly expressed in stomach, C2- and C3-type mRNAs were weakly expressed in intestine. After bacterial challenge, up-regulation was obvious in kidney and spleen for C1-type lysozyme mRNA, while for C2- and C3-type lysozyme obvious increase were observed in stomach and liver, suggesting that C1-type lysozyme may mainly play roles in defense, while C2- and C3-type lysozyme mainly conduct digestive function against bacteria infection. All the three c-type recombinant lysozymes displayed lytic activity against Gram-negative and Gram-positive bacteria. These results indicated that three c-type lysozymes play important roles in the defense of O. aureus against bacteria infections.

Topics: Aeromonas hydrophila; Animals; Cichlids; Cloning, Molecular; DNA, Complementary; Escherichia coli; Fish Proteins; Gene Expression Regulation; Gram-Negative Bacterial Infections; Molecular Sequence Data; Muramidase; Phylogeny; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Sequence Alignment; Sequence Analysis, Protein; Tissue Distribution

The impact of agrichemicals on aquatic vertebrate species has been a matter of increasing concern to researchers and environmentalist. In the present study, we evaluated the effects of a sublethal concentration of atrazine (10% of the LC(50-96 h)), a world-wide used herbicide, on the innate immune system of silver catfish (Rhamdia quelen). A significant reduction on phagocytic index, bacteria agglutination and bactericidal activity of the serum, serum lysozyme and total serum peroxidase activity was observed in fish exposed to atrazine for 24 h. After 10 days exposure to atrazine, only bactericidal activity of the serum, bacteria agglutination and total serum peroxidase activity were significantly reduced. Atrazine had no effect on the natural complement hemolytic activity. Our results demonstrate that atrazine decreases the innate immune response of fingerlings, which might increase its susceptibility to opportunistic pathogens.

Topics: Actinomycetales Infections; Aeromonas hydrophila; Animals; Aquaculture; Atrazine; Catfishes; Complement Hemolytic Activity Assay; Dose-Response Relationship, Drug; Female; Fish Diseases; Gram-Negative Bacterial Infections; Herbicides; Immunity, Innate; Male; Micrococcus luteus; Muramidase; Peroxidase; Water Pollutants, Chemical

The development and duration of immune protection against Aeromonas hydrophila infections with garlic as an immunostimulant in rainbow trout Oncorhynchus mykiss was studied. Rainbow trout fingerlings of 14 g average weight were fed with 0 g (=Control), 0.5 g and 1.0 g of garlic 100 g(-1) of feed for 14 days. Physiological factors, biochemical, immunological, haematological parameters and electrolyte indices were evaluated after a further 14, 21 and 28 days before challenge with Aeromonas hydrophila. Fourteen days after the cessation of feeding with garlic, mortality rates of 12% (relative percent survival [RPS] = 86%) and 16% (RPS = 80%) were recorded in groups which received 0.5 g and 1.0 g of garlic 100 g(-1) of feed, respectively, compared to 84% mortalities in the controls. The corresponding RPS 21 days after ending the feeding regime was 75% and 68, respectively. One week later, the RPS had dropped to 55% and 46% in the groups fed with 0.5 g and 1.0 g garlic 100 g(-1) of feed, respectively.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Blood Cell Count; Diet; Fish Diseases; Garlic; Gram-Negative Bacterial Infections; Muramidase; Oncorhynchus mykiss; Peroxidase; Respiratory Burst

Lysozyme is an enzyme that cleaves the β-1,4-glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, leading to bacterial lysis. Recently, lysozyme has been found to have anti-HIV and anti-cancer properties in mammals. However, most functional analyses were done in vitro using purified or recombinant lysozyme protein. Here, we used RNA interference to silence c-type lysozyme expression in penaeid shrimp, Marsupenaeus japonicus, to analyze the function of lysozyme in vivo. Silencing of lysozyme expression by dsRNA lysozyme (dsLYZ) led to 100% mortality without any artificial bacterial infection in 5 days. Lysozyme deficiency caused the number of hemocytes in hemolymph to decrease from 1.3 × 10(7) to 2.3 × 10(6) cells/ml and caused the number of bacteria to increase from 78 to 764 colony-forming units/ml. Suppression of bacterial growth using oxytetracycline and kanamycin showed improvement in mortality, suggesting that shrimp mortality post- dsLYZ injection can be attributed to bacterial growth in the shrimp hemolymph. The majority of the bacteria, identified by 16 S rRNA analysis, were Gram-negative species such as Vibrio and Pseudomonas. Furthermore, PKH26 staining showed that the dsLYZ-injected shrimp were unable to eliminate non pathogenic Escherichia coli or Staphylococcus aureus in 24 h. These data suggest that c-type lysozyme in shrimp serves to regulate the growth of bacterial communities, particularly Gram-negative bacteria, in the hemolymph.

Topics: Animals; Arthropod Proteins; Gene Expression Regulation, Enzymologic; Gene Silencing; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Hemocytes; Hemolymph; Muramidase; Penaeidae; Staphylococcal Infections; Staphylococcus aureus

To determine the effect of lipopolysaccharide (LPS) for the prevention of infection by Aeromonas hydrophila in rainbow trout (Oncorhynchus mykiss Walbaum) fingerlings.. Rainbow trout fingerlings were fed with 0 mg (= controls), 1.875 mg, 3.75 mg, 7.5 mg and 15 mg of LPS per 100 g of commercial feed for 14 days before experimental challenge with A. hydrophila. The results revealed a reduction in mortalities to 5% in the two lowest doses and 15% in the group, which received 15 mg LPS per 100 g of feed, compared with 45% mortalities in the control. LPS exerted a powerful oxidative burst effect and was a potent mediator of phagocytic, lysozyme, bactericidal and antiprotease activities and total protein. However, whereas there were increases in specific growth rate (SGR), feed conversion ratio (FCR) and protein efficiency ratio (PER) in LPS-treated fish, the data were not significantly (P > 0.05) different.. LPS was effective at preventing disease caused by A. hydrophila and in stimulating the innate immune response of rainbow trout.. The results of this study highlight the role of LPS in fish disease control.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Lipopolysaccharides; Muramidase; Oncorhynchus mykiss; Respiratory Burst

Goldfish, Carassius auratus (47 + or - 3 g, n = 300) were inoculated intramuscularly (50 microl) with Aeromonas hydrophila (1.8 x 10(6) cells ml(-1)). On the 6th day of post-infection the fishes were divided into i) control, without infection fed with normal diet (C), ii) infected fish, fed with normal diet (IU), and infected fishes treated with different doses of iii) 100 mg kg(-1), iv) 200 mg kg(-1), iv) 400 mg kg(-1) and vi) 800 mg kg(-1) mixed herbal extracts supplementation diets. Hematological and immunological parameters were determined on week 1, 2 and 4. In infected goldfish were fed diets containing 100 and 200 mg kg(-1) of mixed herbal extracts supplementation feeds, the white blood cell (WBC) levels significantly increased (P < 0.05) throughout the experimental trial compared to the control. During the experimental period, the red blood cell (RBC) and haemoglobin (Hb) level in goldfish significantly decreased (P < 0.05) when fish fed with 100 and 200 mg kg(-1) of mixed herbal extracts supplementation feeds while it was restored near control when infected fish fed with 400 or 800 mg kg(-1) of herbal extracts supplementation feeds. On the other hand, the haematocrit (Ht) values decline significantly (P < 0.05) in 100, 200 and 400 mg kg(-1) of mixed herbal supplementation feeding groups on weeks 2 and 4 when compared to control group. The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) values almost significantly differ from the control values. The infected goldfish and treated with 100 or 200 mg kg(-1) of herbal supplementation feeds exhibited significantly decline (P < 0.05) in total protein (TP), glucose (GLU) and cholesterol (CHO) levels on week 1-4 whereas it was restored when infected fish fed with 400 or 800 mg kg(-1) of herbal supplementation feeds on week 4. In comparison to untreated control goldfish, the respiratory burst activity and phagocytic activity of blood cells was significantly enhanced in infected fish feeding with 200, 400 and 800 mg kg(-1) of herbal supplementation feeds compared to the control. On the other hand, infected fish fed with all the doses of mixed herbal supplementation feeds, the lysozyme activity was significantly enhanced throughout the experimental period. This study shows that the infected goldfish treated with 400 and 800 mg kg(-1) of herbal supplementation feeds preceding the challenge with live A. hydrophila had 30% and 25% mortality

Topics: Aeromonas hydrophila; Animal Feed; Animals; Blood Cell Count; Diet; Dietary Supplements; Fish Diseases; Goldfish; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Phagocytosis; Plant Extracts; Plants, Medicinal; Respiratory Burst; Time Factors

Lysozyme is an important molecule of innate immune system for the defense against bacterial infections. We identified two genes encoding g-type and c-type lysozymes from grass carp Ctenopharyngodon idellus by the RACE method. The deduced amino acids of both lysozymes possessed typical structural residues and conserved catalytic sites similar to their counterparts across the species. In contrast, there was only 8.6% similarity of amino acid sequence between these two lysozymes. Phylogenetic analyses revealed that these two genes evolved at different rate. C-type lysozyme of grass carp was diverged early in the evolutionary history. Moreover, the expression patterns of these two genes differed. The mRNA levels of both genes were increased after bacterial infection, but the up-regulation of g-type lysozyme was much stronger than that of c-type lysozyme. We also showed that the c-type and g-type recombinant lysozymes possessed different lytic activities against fish bacterial pathogens. These results confirmed that both lysozymes play important roles in the defense of grass carp against bacterial infections. The g-type lysozyme may be induced for the defense against bacterial infections, while c-type lysozyme might be the main molecule for the house-keeping defense under normal conditions. These two types of lysozymes likely use different mechanisms to regulate their expressions.

Topics: Aeromonas hydrophila; Amino Acid Sequence; Animals; Bacteriolysis; Carps; Cloning, Molecular; Evolution, Molecular; Fish Proteins; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacterial Infections; Homeostasis; Immunity, Innate; Molecular Sequence Data; Muramidase; Phylogeny; Sequence Homology, Amino Acid; Species Specificity

Allicin was fed at 0 (= control), 0.5 and 1.0 mL of Allimed liquid 100 g(-1) of feed for 14 days to rainbow trout, Oncorhynchus mykiss (Walbaum), fingerlings before infection with Aeromonas hydrophila with a resultant reduction in mortalities from 80% in the controls to 8% [relative percentage survival (RPS) = 90%] and 0% (RPS = 100%) among the treated fish. Allicin was strongly antibacterial compared to the control, with a minimum inhibitory concentration (MIC) of >400 microL mL(-1) of Allimed liquid. Use of allicin led to a lower number of white blood cells (132.0 +/- 0.4 x 10(3)) compared to 175.0 +/- 0.1 x 10(3) in the controls, but elicited increased phagocytic activity, i.e. a phagocytic value of 39.2% compared to 13.6% in the controls, and serum lysozyme activity, which showed significant (P > 0.05) differences compared to the control at 15 and 30 min after the first reading at 0 min of incubation.

Topics: Aeromonas hydrophila; Animals; Anti-Infective Agents; Disulfides; Enzyme Activation; Fish Diseases; Gram-Negative Bacterial Infections; Leukocyte Count; Microbial Sensitivity Tests; Muramidase; Oncorhynchus mykiss; Phagocytosis; Sulfinic Acids; Superoxides; Time Factors

Feeding rainbow trout, Oncorhynchus mykiss (Walbaum), with 1% lupin, Lupinus perennis, mango, Mangifera indica, or stinging nettle, Urtica dioica, for 14 days led to reductions in mortality after challenge with Aeromonas hydrophila. In addition, there was significant enhancement in serum bactericidal activity, respiratory burst and lysozyme activity in the treatment groups compared to the controls. Use of lupin and mango led to the highest number of red blood and white blood cells in recipient fish, with use of stinging nettle leading to the highest haematocrit and haemoglobin values; the highest value of mean corpuscular volume and haemoglobin was in the control groups and those fed with stinging nettle.

Topics: Aeromonas hydrophila; Animals; Fish Diseases; Food Additives; Gram-Negative Bacterial Infections; Immunity, Innate; Lupinus; Magnoliopsida; Mangifera; Muramidase; Oncorhynchus mykiss; Respiratory Burst; Survival Analysis; Urtica dioica

Parameters of non-specific immune response and level of specific and natural antibodies were investigated after an experimental challenge with Aeromonas hydrophila in genetically different common carp (Cyprinus carpio) families. Ten resistant and ten sensitive families were used for the experiment, which had been selected out of 96 families, based on the results of a preliminary challenge test. Blood samples were collected 12 h, one week and 21 days following the challenge. Phagocytic and respiratory burst activities of phagocytic cells, lysozyme activity of the blood plasma were determined. Level of specific antibodies against A. hydrophila and level of natural antibodies were measured in the samples taken on the 28th day. Non-infected fish from resistant and sensitive families were used as controls. Significant differences of phagocytic and lysozyme activities were measured between the challenged resistant and sensitive families. The level of specific antibodies between the same families was also found to be significantly different. There were no significant differences of the studied parameters between the control groups. Based on our results, phagocytic activity of leukocytes, plasma lysozyme activity and specific antibody titre were found to be higher in the resistant families than in the sensitive ones following infection with A. hydrophila.

Topics: Aeromonas hydrophila; Animals; Antibodies, Bacterial; Carps; Female; Fish Diseases; Genetic Predisposition to Disease; Gram-Negative Bacterial Infections; Leukocytes, Mononuclear; Male; Muramidase; Phagocytosis; Respiratory Burst; Selection, Genetic

The present study evaluated the efficacy of dietary doses of Withania somnifera (Ashwagandha) root powder on immunological parameters and disease resistance against the bacterial pathogen Aeromonas hydrophila infections in Indian major carp, Labeo rohita fingerlings. Fishes were fed with dry diet containing 0 gkg(-1) (control), 1 gkg(-1) (T(1)), 2 gkg(-1) (T(2)) and 3 gkg(-1) (T(3)) W. somnifera root powder for 42 days. Immunological (NBT level, Phagocytic activity, total immunoglobulin and lysozyme activity) parameters of fishes were examined at 0 days, 14 days, 28 days and 42 days of feeding. Fishes were challenged with A. hydrophila 42 days post feeding and mortalities (%) were recorded over 14 days post-infection. The results demonstrate that fishes fed with W. somnifera root showed enhanced NBT level, Phagocytic activity, total Immunoglobulin level and lysozyme activity (p<0.05) compared with the control group. The survivability was higher in experimental diets than the control group. Dietary W. somnifera at the level of 2 gkg(-1) showed significantly (P<0.05) higher protection (RPS 42.85+/-0.65%) against A. hydrophila infection than control. The results suggest that the W. somnifera root powder have a stimulatory effect on immunological parameters and increases disease resistance in L. rohita fingerlings against A. hydrophila infection.

Topics: Aeromonas hydrophila; Animals; Cyprinidae; Diet; Fish Diseases; Food Additives; Gram-Negative Bacterial Infections; Immunity, Innate; Immunoglobulins; Muramidase; Nitroblue Tetrazolium; Phagocytosis; Plant Roots; Survival Analysis; Withania

The effect of traditional Chinese medicine (TCM) formulated from propolis and Herba Epimedii extracts at the ratio of 3:1 (w/w) on non-specific immune response of Chinese sucker (Myxocyprinus asiaticus) was investigated. Fish were fed diets containing 0 (control), 0.1%, 0.5% or 1.0% TCM extracts for five weeks. The respiratory burst and phagocytic activities of blood leukocytes, lysozyme and natural haemolytic complement activities in plasma were measured weekly. After five weeks of feeding, fish were infected with Aeromonas hydrophila and mortalities were recorded. Results of this study showed that feeding Chinese sucker with different dosage of TCM extracts stimulated respiratory burst activity, phagocytosis of phagocytic cells in blood and lysozyme activity in plasma. They had no effect on plasma natural haemolytic complement activity. All dosage of treated groups showed reduced mortality following A. hydrophila infection. Feed containing 0.5% TCM extracts was the most effective with the mortality of the fish significantly reduced by 35% compared to the control. The results indicate that propolis and Herba Epimedii extracts in combination enhances the non-specific immune response and disease resistance of Chinese sucker against A. hydrophila.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Complement System Proteins; Cypriniformes; Drugs, Chinese Herbal; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Phagocytosis; Plant Extracts; Propolis; Respiratory Burst

The study evaluated the effect of dietary doses of Euglena viridis on the immune response and disease resistance of Labeo rohita fingerlings against infection with the bacterial pathogen Aeromonas hydrophila. L. rohita fingerlings were fed with diet containing 0 (Control), 0.1 g, 0.5 g, 1.0 g Euglena powder kg(-1) dry diet for 90 days. Biochemical (serum total protein, albumin, globulin, albumin:globulin ratio), haematological (WBC, RBC, haemoglobin content) and immunological (superoxide anion production, lysozyme, serum bactericidal activity) parameters of fish were examined after 30, 60 and 90 days of feeding. Fish were challenged with A. hydrophila 90 days post-feeding and mortalities were recorded over 10 days post-infection. The results demonstrate that fish fed with Euglena showed increased levels of superoxide anion production, lysozyme, serum bactericidal activity, serum protein and albumin (P < 0.05) compared with the control group. Following challenge with A. hydrophila less survivability was observed in the control group (56.65%) than the group fed the experimental diets. The group fed 0.5 g Euglena kg(-1) dry diet showed the highest percentage survival (75%). These results indicate that Euglena stimulates the immunity and makes L. rohita more resistant to A. hydrophila infection.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Blood Proteins; Cyprinidae; Erythrocyte Count; Euglena; Fish Diseases; Gram-Negative Bacterial Infections; Hemoglobins; Leukocyte Count; Muramidase; Superoxides; Survival Analysis

Propolis, a resinous substance collected by Apis mellifera bees from various plant sources and mixed with secreted beeswax, is a multifunctional material used by bees in the construction, maintenance, and protection of their hives. The collected propolis sample, from High Egypt, was dark-green with olive-odor. The minimal inhibition concentration (MIC) of propolis-ethanolic-extract, against Aeromonas hydrophila, was 80 microg Propolis-ethanolic-extract and crude propolis (1%) were added to artificial basal diet with (30% crude protein) to evaluate their efficacy on the fish growth-performance, immunostimulation and resistance to A. hydrophila. Two hundred and twenty-five Oreochromis niloticus (8 +/- 0.45 g/fish) were divided into three equal treatments (T) of triplet replicates. The fish of T(1) were fed on basal diet (control). The fish of T(2) were given the basal diet, containing propolis-ethanolic-extract. The fish of T(3) were given the basal diet containing crude propolis for 28 day. The fish were intraperitoneally challenged by A. hydrophila (0.2 x 10(7) cells ml(-1)) at the end of the feeding period and kept for 15 more days. The best growth rate and feed conversion ratio were obtained with T(2.) The increase in the average daily gain, specific growth rate and feed efficiency ratio were highly significances in T(2) followed by T(3) when compared with the control group. The HCT-level and monocyte-counts were increased (T(2)). No significant change, in the large lymphocytic-count was found among the three treatments (28-27-28%), while the neutrophil-count was significantly decreased (7%) with T(2) and increased (13.11%) with the control. A significant increase in serum lysozyme and serum bactericidal activities was found with T(2). The RLP against A. hydrophila was high with T(2) and T(3). The propolis-ethanolic-extract enhanced the growth, immunity and resistance of O. niloticus against A. hydrophila more than the crude propolis.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Anti-Infective Agents; Cichlids; Fish Diseases; Gram-Negative Bacterial Infections; Hematocrit; Immunity, Innate; Leukocyte Count; Microbial Sensitivity Tests; Muramidase; Propolis; Time Factors

This study reports the effect of aqueous, ethanol and methanol triherbal solvent extract from Azadirachta indica, Ocimum sanctum and Curcuma longa on innate immune mechanisms such as phagocytosis activity, respiratory burst activity, alternative complement activity and lysozyme activity and disease resistance in goldfish (Carassius auratus) against Aeromonas hydrophila. Fish were intraperitoneally injected with different doses of 0, 5, 50 and 100 mg kg(-1) body weight of each triherbal solvent extracts. The functional immunity in terms of percentage mortality and Relative Percent Survival (RPS) and innate immune response was assessed on week 1, 2 and 4 by challenging with live A. hydrophila (1 x 10(7) cells ml(-1)). All the chosen innate immune parameters were enhanced in the ethanol and methanol triherbal solvent extract treatment after week 2. However, the aqueous triherbal extract was enhanced only after week 4. The ethanol and methanol triherbal solvent extracts administration preceding the challenge with live A. hydrophila decreased the percentage mortality in the experimental groups with the consequence increase in RPS values. The study indicates that all the doses of ethanol or methanol triberbal solvent extracts could be positively influence the immune response and protect the heath status of goldfish against A. hydrophila infection.

Topics: Aeromonas hydrophila; Animals; Azadirachta; Complement System Proteins; Curcuma; Fish Diseases; Goldfish; Gram-Negative Bacterial Infections; Immunity, Innate; Magnoliopsida; Muramidase; Ocimum; Phagocytosis; Plant Extracts; Respiratory Burst

The present experiment was carried out to study the effect of different dosages of beta-glucan suspension derived from barley on the innate immune response and disease resistance of Anabas testudineus spawns against infection caused by Aeromonas hydrophila. Four different dosages of beta-glucan suspension in phosphate buffered saline at the rate of 0, 5, 10, 15 mg l(-1) were taken and 8 days old spawn were exposed for 2 h and 3 h. The cell suspension of spawn was assayed for total protein, acid phosphatase activity, lysozyme activity, bactericidal and NBT. Further, the spawns were challenged with 3 x 10(5) cells ml(-1) of A. hydrophila and survivability percentage and immunological parameters were assayed upto day 7. On day 7, most of the immunological parameters such as lysozyme activity, bactericidal activity and NBT activity were significantly enhanced after exposing the fish to all the concentrations of beta-glucan. Challenge study indicated least mortality in the group of spawns immersed in 15 mg l(-1) beta-glucan suspension for 3 h. Thus, 3 h exposure to beta-glucan suspension could reduce the mortality and increase the immunity of A. testudineus spawns.

Topics: Acid Phosphatase; Aeromonas hydrophila; Animals; beta-Glucans; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Perches; Superoxides; Survival Analysis

Garlic, Allium sativum, which was fed at 0, 0.05, 0.1, 0.5 and 1.0 g per 100 g of feed for 14 days to rainbow trout, Oncorhynchus mykiss (Walbaum), led to control of experimental infection with Aeromonas hydrophila. At doses of 0.5 and 1.0 g garlic per 100 g of feed, there was a reduction in mortalities to 4% compared with the controls (88%). Moreover, there was a significant increase in growth, feed conversion and protein efficiency. There was stimulation of the number of erythrocytes and leucocytes, a significantly higher haematocrit, enhancement of phagocytic activity, respiratory burst, lysozyme, anti-protease and bactericidal activities following feeding with garlic.

Topics: Aeromonas hydrophila; Animals; Blood Cell Count; Diet; Dietary Supplements; Fish Diseases; Garlic; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Oncorhynchus mykiss; Phagocytosis; Random Allocation; Superoxides

Ginger, Zingiber officinale, which was fed at 0, 0.05, 0.1, 0.5 and 1.0 g per 100 g of feed for 14 days to rainbow trout, Oncorhynchus mykiss (Walbaum), led to control of experimental infection with Aeromonas hydrophila. At 0.5 g ginger per 100 g of feed, there was a reduction in mortalities to 0% compared with the controls (64%). Moreover, there was a significant increase in growth, feed conversion and protein efficiency. There was proliferation in the number of neutrophils, macrophages and lymphocytes, and enhanced phagocytic, respiratory burst, lysozyme, bactericidal and anti-protease activities compared with the controls.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Blood Cell Count; Colony Count, Microbial; Diet; Dietary Supplements; Fish Diseases; Gram-Negative Bacterial Infections; Hemoglobins; Immunity, Innate; Muramidase; Oncorhynchus mykiss; Phagocytosis; Superoxides; Zingiber officinale

The present study was performed to investigate the immunostimulatory effects of Korean mistletoe extract (KM-110; Viscum album Coloratum) on the non-specific immune response and protection against Aeromonas hydrophila infection in Japanese eel (Anguilla japonica). Eels were fed under 4 regimes, 0%, 0.1%, 0.5% and 1.0% KM-110 mixed diet. On day 14 after feeding, 15 fish from each group were injected i.p. with live A. hydrophila (3 x 10(6)CFU) and the remaining unchallenged fish from each group were used to study the innate immune response. On 14 days post-infection, the total survival rates were 26.6% in control, and 33.3%, 66.6% and 80% in 0.1%, 0.5% and 1% KM-110-treated groups, respectively. The maximum lysozyme activity was observed in the 1% KM-110-treated group. There was no significant difference of lysozyme activity between 0.1% and 0.5% KM-110 group. Superoxide anion (O(2)(-)) production was significantly (p<0.05) augmented in the 0.5% and 1% KM-110 groups compared to the control and 0.1% KM-110 group. No significant difference of (O(2)(-) production was found between 0.5% and 1% KM-110 group. Likewise, there was a significant increase in phagocytic activity in the 0.5% KM-110 group compared with the 0.1% group (p<0.05), but no significant difference between the 0.5% and the 1% KM-110 group indicating that 0.5% KM-110 concentration is suitable for stimulating maximum phagocytic activity resulting in a high amount of ROI production. Considering the present results, KM-110 could be utilized as a promising immunostimulating substance for a diet in aquaculture.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Anguilla; Animals; Diet; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Cellular; Immunity, Innate; Muramidase; Phagocytosis; Plant Extracts; Reactive Oxygen Species; Survival Analysis; Viscum album

Lysozymes are key proteins of the innate immune system against bacterial infections. In this study we report the molecular cloning and characterization of the c-type and g-type lysozymes in brill (Scophthalmus rhombus). Catalytic and other conserved residues required for functionality were identified. Phylogenetic analysis revealed distinct evolutionary histories for each lysozyme type. Expression profiles of both lysozyme genes were studied in juvenile tissues using a real-time PCR approach. c-Type lysozyme was expressed mainly in stomach and liver, whereas the g-type was detected in all tissues with highest mRNA levels observed in the spleen. Induction experiments revealed that g-type transcripts increased significantly in head kidney after lipopolysaccharide (25- and 23-fold at 12 and 24h, respectively) and Photobacterium damselae subsp. piscicida (17-fold at 24h) treatments. In contrast, no induction was observed for c-type lysozyme. All these data suggest that g-type lysozyme is involved in the response against bacterial infections, whereas c-type lysozyme may also play a role in digestion.

Topics: Amino Acid Sequence; Animals; Fish Diseases; Flatfishes; Gene Expression Regulation, Enzymologic; Gram-Negative Bacterial Infections; Lipopolysaccharides; Molecular Sequence Data; Muramidase; Photobacterium; Phylogeny; Sequence Alignment

A preliminary study with a 75days feeding trial was conducted to study the immunomodulatory effect of microbial levan on Cyprinus carpio juveniles. Five purified isonitrogenous and isocaloric diets with graded levels of levan, namely (T(1)) 0.1% levan, (T(2)) 0.2% levan, (T(3)) 0.5% levan, (T(4)) 1.0% levan, and a control group without levan were fed to five groups of fishes in triplicate. The total erythrocyte count and haemoglobin content was significantly (p<0.05) enhanced in the T(3) group, though the total leucocyte counts, total protein and A/G ratio did not show any significant difference (p>0.05) when compared with the control group. The respiratory burst activity (NBT) of blood phagocytes and lysozyme activity was also highest in T(3) group. The relative survival percentage after challenge with Aeromonas hydrophila was highest (100%) in the T(3) group followed by 83.33% and 66.67% in the T(2) and T(4) groups, respectively. This suggests that microbial levan at 0.5% can be used as a dietary immunostimulant for C. carpio juveniles. This is the first report on microbial levan having an immunomodulatory effect on C. carpio.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animal Feed; Animals; Carps; Diet; Erythrocytes; Fish Diseases; Fructans; Gram-Negative Bacterial Infections; Hemoglobins; Immunity, Innate; Muramidase; Phagocytes; Random Allocation; Respiratory Burst; Survival Analysis; Time Factors

The study evaluated the efficacy of dietary doses of Mangifera indica (mango) kernel on the immune response and disease resistance of Labeo rohita fingerlings against the bacterial pathogen Aeromonas hydrophila infections in. L. rohita fingerlings fed diet containing 0 (Control), 1g, 5 g, 10 g mango kernel kg(-1) dry diet for 60 days. Biochemical (serum total protein, albumin, globulin, albumin:globulin ratio, blood glucose), haematological (WBC, RBC, haemoglobin content) and immunological (superoxide anion production, lysozyme, serum bactericidal activity) parameters of fish were examined at 20, 40 and 60 days of feeding. Fish were challenged with A. hydrophila 60 days post feeding and mortalities were recorded over 10 days post-infection. The results demonstrate that fish fed with mango kernel showed enhanced superoxide anion production, lysozyme, serum bactericidal, serum protein, albumin (P<0.05) compared with the control group. The mortality (%) was recorded up to 10 th day post-challenge. Less survivability was observed in control group (50%) up to day 10 after infection. The survivability was higher in experimental diets. The group fed 5 g kernel kg(-1) dry diet showed highest percentage survival (98%). These results indicate that mango kernel stimulates the immunity and makes L. rohita more resistant to A. hydrophila infection.

Topics: Aeromonas hydrophila; Analysis of Variance; Animals; Blood Chemical Analysis; Blood Proteins; Cyprinidae; Fish Diseases; Food Additives; Gram-Negative Bacterial Infections; Immunity, Innate; Mangifera; Muramidase; Seeds; Serum Albumin; Superoxides; Survival Analysis; Time Factors

Goldfish (Carassius auratus) were exposed to a mixture of herbicides, namely atrazine, simazine, diuron, and isoproturon (ASDI) at a cumulative concentration of 50microg/l for 12 weeks. Control fish and exposed fish were sampled at 4, 8 and 12 weeks of exposure to observe the combined impact of herbicides on non-specific and specific mechanisms of immunity and antioxidant defenses. The antioxidant defenses were evaluated in haemopoietic organs and liver. ASDI-induced stress was reflected as a significant induction of superoxide (O(2)(-)) production in phagocytic cells of head kidney and spleen. In addition, plasma lysozyme activity was consistently high in the treatment group. Specific immunity was assessed by antibody titre responses following immunization of the fish with sheep red blood cells (SRBCs). Antibody titre was reduced throughout the period of observation in ASDI-treated fish. This reduction was found to be significant at week 4 (p<0.05). Herbicide-exposed fish showed reduced resistance against pathogen invasion but remarkable enhancement in lysozyme activity. The ASDI-induced oxidative stress in spleen, kidney and liver was reflected as a change in the antioxidant enzyme activities in these vital organs of fish. Our data indicate that herbicides at concentrations present in water bodies in Europe cause immune suppression in goldfish.

Topics: Aeromonas hydrophila; Animals; Antibodies; Antibody Formation; Biomarkers; Catalase; Erythrocytes; Female; Goldfish; Gram-Negative Bacterial Infections; Herbicides; Immunization; Male; Muramidase; Phagocytes; Superoxide Dismutase; Superoxides; Time Factors; Water Pollutants, Chemical

A 60 days experiment was conducted to study the effect of dietary gelatinized (G) and non-gelatinized (NG) starch on immunomodulation of Labeo rohita juveniles. Two hundred and thirty four juveniles (av. wt. 2.53+/-0.04) were randomly distributed in six treatment groups with each of three replicates. Six semi-purified diets containing NG and G corn starch, each at six levels of inclusion (0, 20, 40, 60, 80, 100) were prepared viz., T(1) (100% NG, 0% G starch), T(2) (80% NG, 20% G starch), T(3) (60% NG, 40% G starch), T(4) (40% NG, 60% G starch), T(5) (20% NG, 80% G starch) and T(6) (0% NG, 100% G starch). After a feeding period of 60 days, the juveniles were challenged with Aeromonas hydrophila to study their immunomodulation due to feeding of G and NG starch. RBC and haemoglobin content were significantly (P<0.05) reduced due to bacterial challenge, but dietary starch (G/NG starch) had no effect on it. G:NG starch ratio in the feed had significant effect on total leukocyte count during pre- and post-challenge periods. The leukocyte count concomitantly increased with the increased level of G starch in the diet. Highest albumin/globulin (A/G) ratio was recorded in T6 group (100% G starch) and lowest in T1 group (100% NG starch) group followed by T2 group both in pre- and post-challenge periods. NBT, lysozyme activity, total protein and globulin content were highest in T2 group (80% NG, 20% G starch) both in pre- and post-challenge periods. After challenge with A. hydrophila, the highest survival was recorded in T2 group, whereas lowest survival was recorded in T6 group. Conclusively high level of G starch was found to be immunosuppressive in Labeo rohita juveniles and NG:G starch ratio of 80:20 seems to be optimum for promoting growth and protecting immunity in L. rohita juveniles.

Topics: Aeromonas hydrophila; Animals; Blood Cell Count; Blood Proteins; Body Weight; Carps; Cholesterol; Dietary Carbohydrates; Fish Diseases; Gelatin; Gram-Negative Bacterial Infections; Hemoglobins; Immunologic Factors; Muramidase; Random Allocation; Respiratory Burst; Starch; Survival Analysis; Triglycerides

The purpose of this study was to determine the effect of water and hexane soluble fractions of the Indian medicinal plant, Solanum trilobatum on the nonspecific immune mechanisms and disease resistance in Oreochromis mossambicus. Fish were intraperitoneally injected with different doses of 0, 4, 40 or 400 mg kg(-1) body weight of water or hexane soluble fraction. The nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, reactive oxygen species production and reactive nitrogen species production by peripheral blood leucocytes. The functional immunity in terms of percentage mortality and Relative Percent Survival (RPS) was assessed by a challenge with live Aeromonas hydrophila. Almost all the doses of both water and hexane soluble fractions enhanced the serum lysozyme activity. All the doses of water soluble fraction significantly enhanced the ROS production on most of the days tested. In hexane soluble fraction treated groups, the enhancement in the ROS production was observed at least on 2 days. All the doses of water soluble fraction significantly enhanced the production of RNS only on one day. The RNS production was enhanced significantly only in the group treated with 40 mg kg(-1) of hexane fraction. The leaf fraction administration preceding the challenge with live A. hydrophila, decreased the percentage mortality in the experimental group with the consequent increase in RPS values. This preliminary study indicates that S. trilobatum could be used to promote the health status of fish in intensive finfish aquaculture.

Topics: Aeromonas hydrophila; Animals; Aquaculture; Fish Diseases; Gram-Negative Bacterial Infections; Male; Muramidase; Plant Extracts; Plant Leaves; Plants, Medicinal; Reactive Nitrogen Species; Reactive Oxygen Species; Solanum; Tilapia

Achyranthes aspera seed was incorporated in the diets (at 0.01%, 0.1% and 0.5%) of Labeo rohita, rohu fingerlings (3.0+/-0.4 g). After 2 weeks, the fish were immunized with heat-killed Aeromonas hydrophila, and after a further 2 weeks the rohu were experimentally infected with Aeromonas hydrophila (ATCC 49140). After 7 days blood and serum were sampled to determine superoxide anion production, bactericidal activity, lysozyme, serum protein, albumin, globulin, serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and alkaline phosphatase (ALP). Superoxide anion production, serum bactericidal activity, lysozyme, ALP, serum protein, albumin:globulin ratio (A/G) were enhanced in Achyranthes treated groups compared to the control group. SGOT and SGPT levels were elevated in control group, but in Achyranthes treated groups the levels were similar to the uninfected-control group. Higher cumulative mortalities were observed in the control group (77%) up to day-9 after infection. This gradually decreased with increasing dose of Achyranthes, 66% mortality in 0.01% group, 57% mortality in 0.1% group and 28% mortality in 0.5% group. These results indicate that Achyranthes aspera stimulates immunity and increases resistance to infection in L. rohita.

Topics: Achyranthes; Aeromonas hydrophila; Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Blood Proteins; Cyprinidae; Dose-Response Relationship, Drug; Fish Diseases; Gram-Negative Bacterial Infections; Muramidase; Phytotherapy; Seeds; Serum Albumin; Serum Globulins; Superoxides

The purpose of this study was to determine if multiple injections of different dosages of beta-glucan derived from barley would enhance the immune response and disease resistance against infections due to opportunistic pathogens Aeromonas hydrophila and Edwardsiella tarda in Labeo rohita fingerlings. Hence, four different dosages of beta-glucan suspension in phosphate-buffered saline at the rate of 0, 5, 10, 15 mg kg(-1) body weight of fish were injected intraperitoneally to the fingerlings of Labeo rohita at two-week intervals for four times. After every two-week interval different serum biochemical, haematological and immunological parameters of fish were evaluated. At the end of immunostimulation trial of 56 days, fish were divided into four subgroups under each major treatment group for challenge through i.p injection and bath immersion with two pathogens, A. hydrophila and E. tarda. The mortality (%) and agglutinating antibody titre was recoded on 28th day post challenge. Most of the immune parameters such as leucocyte count, phagocytic ratio, phagocytic index, lysozyme activity, complement activity, serum bactericidal activity were significantly (P < 0.05) enhanced on 42 days after three i.p injections of 10 mg of beta-glucan kg(-1) body wt. Challenge study indicated least mortality in the group of fishes injected with medium dose of 10 mg of beta-glucan kg(-1) body wt. four times. Multiple injections of beta-glucan might have maintained the activation of phagocytic cells for a long period which in turn would lead to long-term protection in fishes. Thus, injections of 10 mg of beta-glucan kg(-1) body wt. for three times can be advocated to enhance the immune response of fish species under aquaculture.

Topics: Aeromonas hydrophila; Animals; beta-Glucans; Blood Chemical Analysis; Cyprinidae; Dose-Response Relationship, Drug; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Injections, Intraperitoneal; Leukocytes; Linear Models; Muramidase; Phagocytosis; Superoxides

The purpose of this study was to determine if injections of different dosages of tuftsin would enhance the immune response and disease resistance against the infections due to the opportunistic pathogens Aeromonas hydrophila and Edwardsiella tarda in Labeo rohita fingerlings. Hence, four different dosages of tuftsin in PBS suspension at the rate of 0, 5, 10, 15 mg kg(-1) body weight of fish were injected intraperitoneally to the fingerlings of L. rohita at 2-week intervals for four times. After every 2-week interval, different serum biochemical, haematological and immunological parameters of fish were evaluated. Biochemical and haematological parameters including serum total protein content, albumin content, globulin content, albulin:globulin ratio, glucose content, leucocyte counts etc.; cellular immune parameters including superoxide anion production, phagocytic activities, lymphokine production index etc.; humoral immune parameters including lysozyme activity, complement activity, serum bactericidal activity etc., in the fish were evaluated after every 2-week interval. After 56 days, fish were divided into two subgroups under each major treatment group for challenge with two pathogens A. hydrophila and E. tarda. The mortality (%) and agglutinating antibody titre was recorded on 28th day post challenge. Most of the immune parameters including leucocyte count, phagocytic ratio, phagocytic index, lysozyme activity, complement activity, and serum bactericidal activity were significantly (p

Topics: Aeromonas hydrophila; Agglutination Tests; Animals; Blood Glucose; Blood Proteins; Carps; Complement System Proteins; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Immunologic Factors; Injections, Intraperitoneal; Leukocyte Count; Lymphokines; Muramidase; Phagocytosis; Serum Bactericidal Test; Superoxides; Time Factors; Tuftsin

This study investigated the effects of short and prolonged administration of a yeast beta-glucan on non-specific immune parameters, growth rate and the disease resistance of Asian catfish, Clarias batrachus. Fish fed with a basal diet (control) and test diet (basal diet supplemented with 0.1% glucan) for 1, 2 and 3 weeks were assayed for superoxide production, serum myeloperoxidase (MPO) content, natural haemagglutinin level, complement and lysozyme activities. Fish were weighed at weekly intervals and specific growth rate (SGR, % increase in body weight per day) was determined. After each week, fish were challenged with Aeromonas hydrophila to measure the level of protection. Results showed that glucan administration at 0.1% in feed, significantly (P<0.05) enhanced MPO and lysozyme levels, superoxide production, haemagglutination titre and level of protection against A. hydrophila challenge, irrespective of length of exposure. The alternative complement activity and SGR were not affected by the dietary supplementation of yeast glucan. As glucan feeding at 0.1% for 1 week is able to enhance the non-specific immunity and disease resistance of catfish efficiently, short-term feeding might be used in farmed catfish diets to enhance disease resistance.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animal Feed; Animals; beta-Glucans; Catfishes; Complement System Proteins; Dietary Supplements; Fish Diseases; Gram-Negative Bacterial Infections; Hemagglutination; Immunity, Innate; Muramidase; Nitroblue Tetrazolium; Peroxidase; Random Allocation; Respiratory Burst; Time Factors

The objective of this study was to investigate the effect of chronic exposure to sublethal concentrations of hexavalent chromium (K2Cr2O7) on the immune response and disease resistance of Oreochromis mossambicus (Peters) to bacterial Aeromonas hydrophila infection. Fish (45 to 50 g) were exposed to 0.005, 0.05, 0.5, and 5 mg l(-1) [0.01, 0.1, 1, and 10% LC50, respectively] of hexavalent chromium Cr (VI) for 28 d. The specific immune response was assessed by antibody response to A. hydrophila by bacterial agglutination assay, and to sheep red blood cells (SRBC) by plaque forming cell (PFC) assay. In addition, nonspecific immune mechanisms were assessed by serum lysozyme activity and reactive nitrogen intermediates, the latter in terms of nitric oxide (NO) production by peripheral blood leucocytes. Overall immunity was assessed by disease resistance against live virulent A. hydrophila. The study clearly indicated that chronic exposure of fish to 0.5 and 5 mg l(-1) of chromium (VI) decreased both nonspecific and specific parameters of the immune system, which resulted in a lower disease resistance to A. hydrophila. Interestingly, 0.05 mg l(-1) of Cr (VI) enhanced disease resistance and both nonspecific and specific immune responses to A. hydrophila. Our study revealed a concentration-dependent modulation of the immune system by chromium (VI), as demonstrated by suppressive or stimulatory effects on lymphocytes, lysozyme, phagocytic killing mechanisms, and disease resistance in O. mossambicus.

Topics: Aeromonas hydrophila; Agglutination Tests; Animals; Chromium; Environmental Exposure; Fish Diseases; Gram-Negative Bacterial Infections; Hemolytic Plaque Technique; Immunity, Innate; Male; Muramidase; Nitric Oxide; Tilapia; Time Factors

Carnobacterium maltaromaticum B26 and Carnobacterium divergens B33, which were isolated from the intestine of healthy rainbow trout (Oncorhynchus mykiss, Walbaum), were selected as being potentially useful as probiotics with effectiveness against Aeromonas salmonicida and Yersinia ruckeri. Thus, rainbow trout administered with feed supplemented with B26 or B33 dosed at >10(7) cells g(-1) feed conferred protection against challenge with virulent cultures of the pathogens. Moreover, both cultures persisted in the gut for up to 3 weeks after administration. The cultures enhanced the cellular and humoral immune responses. Specifically, fish fed with B26 demonstrated significantly increased phagocytic activity of the head kidney macrophages, whereas the use of B33 led to significant increases in respiratory burst and serum lysozyme activity. Also, the gut mucosal lysozyme activity for fish fed with both cultures was statistically higher than the controls.

Topics: Aeromonas; Animals; Bacteria; Blood Cell Count; DNA Primers; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Intestines; Muramidase; Oncorhynchus mykiss; Phagocytosis; Probiotics; Respiratory Burst; Survival Analysis; Time Factors; Trypsin; Yersinia; Yersinia Infections

To examine the immunological impacts of polychlorinated biphenyls (PCBs) in an environmentally relevant way, we orally contaminated Arctic charr (Salvelinus alpinus) with Aroclor 1254. After contamination, fish were either fed (0 and 100 mg Aroclor 1254 kg(-1) fish wt) or fasted (0, 1, 10, and 100 mg kg(-1)) to mimic cycles of feeding-fasting experienced by Arctic animals. After four months, PCB concentrations in muscle were the same in fasted and fed fish; however, PCBs in kidneys of fed fish were 33 to 50% of those in fasted fish. Arctic charr were exposed to Aeromonas salmonicida, the bacteria responsible for furunculosis, by cohabitation with infected conspecifics. Fasted fish had a significant trend toward lower survival with higher dose of PCBs--from 68% in controls to 48% in treatment involving 100 mg kg(-1). Independent of PCB contamination, fed fish had the lowest survival; we attribute this to stress associated with establishing and maintaining feeding hierarchies. A significant decrease in the activity of lysozyme was observed in skin mucus, as was hemagglutination ability of a putative rhamnose lectin in fasted, but not in fed, PCB-treated fish. These results demonstrate the immunosuppressive effects of PCBs on Arctic charr, and they illustrate the importance of considering environmentally relevant nutritional status in ecotoxicological studies.

Topics: Aeromonas salmonicida; Animals; Chlorodiphenyl (54% Chlorine); Food Deprivation; Gram-Negative Bacterial Infections; Immunity, Innate; Kidney; Lectins; Muramidase; Muscle, Skeletal; Salmonidae; Water Pollutants, Chemical

Cyclophosphamide (CYP), a multifunctional alkylating agent is known as a potent immunosuppressor in endotherms. Here, an experiment was conducted in an ectothermic Asian catfish species, Clarias batrachus to investigate its effect on non-specific and specific immunity as well as disease resistance against a common bacterial pathogen challenge. CYP was intraperitoneally injected for 3 consecutive days at a rate of 200 mgkg(-1) body weight. After 72 h post-injection, control and CYP-treated fish were screened for superoxide production through nitroblue tetrazolium (NBT) assay, myeloperoxidase (MPO) activity, packed cell volume (PCV) and total protein, lysozyme, alternative complement activity (ACH50) and natural haemagglutinin titre as a measure of non-specific immunity level as well as disease resistance against Aeromonas hydrophila challenge to vaccinated and unvaccinated fish. To study the effect on specific immunity, CYP was injected thrice at an interval of 7 days after bacterin injection and serum antibody titre was measured by bacterial agglutination titre assay. The results showed a significant (P<0.05) decrease in NBT and MPO activities, and percent survival against A. hydrophila challenge (both in unvaccinated and vaccinated fish) in CYP-treated fish, when compared to control fish. The above results support the immunosuppressive action of CYP in freshwater catfish, C. batrachus. The neutrophil activities, as measured through superoxide production and myeloperoxidase levels, might be important contributors during A. hydrophila infection and that treatment with CYP reduces phagocytic killing power and inhibits resistance against aeromoniasis.

Topics: Aeromonas hydrophila; Animals; Blood Glucose; Catfishes; Complement Pathway, Alternative; Cyclophosphamide; Fish Diseases; Gram-Negative Bacterial Infections; Hematocrit; Hydrocortisone; Immunity, Innate; Immunosuppression Therapy; Injections, Intraperitoneal; Muramidase; Nitroblue Tetrazolium; Peroxidase; Proteins; Superoxides; Survival Analysis

Sea bream broodstock were immunised 1 or 2 months before spawning with a novel photobacteriosis vaccine. Sixty-seven-day-old larvae (mean weight 22.3 mg) originating from immunised and non-immunised parents were experimentally infected with the Photobacterium damsela subsp. piscicida (Phdp). Larvae from immunised fish showed delayed onset and lower mortality (66.67%) compared with larvae from control fish (80%). Eighty-nine-day-old larvae (mean weight 162.2 mg) from both groups were bath vaccinated with Phdp and Escherichia coli lipopolysaccharides (LPS) and larval samples were collected for measurement of humoral parameters. Larvae vaccinated with Phdp and LPS showed significantly higher anti-protease activity, lysozyme activity and total immunoglobulin compared to the controls. One-hundred-and-twenty-day-old larvae (mean weight 297.85 mg) from both parental groups were challenged with (LD70) virulent Phdp bacterial cells. Vaccinated larvae from both groups showed significantly less mortality compared to the respective controls. The RPS values of larvae from immunised parents vaccinated with Phdp and LPS was 95.83% and 72.22%, respectively. The RPS values of larvae from non-immunised parents vaccinated with Phdp and LPS was 62.5% and 70.83%, respectively. Results are discussed with respect to the beneficial effect of broodstock immunisation prior to spawning and the immunisation of larvae on their survival against photobacteriosis.

Topics: Analysis of Variance; Animals; Antibody Formation; Aquaculture; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Gram-Negative Bacterial Infections; Hemagglutination Tests; Immunoglobulins; Larva; Lipopolysaccharides; Muramidase; Photobacterium; Sea Bream; Vaccination

Aeromonas bestiarum is one of the causal agents of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) in fish. Infections of the bacterium is an increasing problem in commercial carp (Cyprinus carpio L.) farmed in Poland. Non-specific immune response of the fish, vaccinated with oil-emulsified experimental vaccines containing formalin killed whole cells (WCs), formalin killed whole culture (WCt) or crude LPS (50 or 1250 microg per fish) were studied on days 7 and 30 after vaccination. Fish vaccinated for 30 days were challenged with the pathogen and mortalities recorded over 14 days. The cumulative mortalities were 10%, 0%, 20% and 20% in WCs, WCt, LPS-1250 and LPS-50 groups, respectively, whereas 70% fish died in the control group. Vaccinated fish showed significant increase of phagocytic activity (PA) and phagocytic index (PI). The total serum Ig (TSIg) level was significantly higher in most vaccinated fish groups than in control. Moreover, WCs and WCt induced significant increase of mucus lysozyme level in vaccinated fish.

Topics: Aeromonas; Animals; Bacterial Vaccines; Carps; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Immunoglobulins; Lipopolysaccharides; Muramidase; Phagocytosis; Poland

An auxotrophic aroA mutant of the Aeromonas hydrophila AG2 strain is a live attenuated vaccine against A. hydrophila infection in rainbow trout (Oncorhynchus mykiss). The protection conferred by the live attenuated vaccine against A. salmonicida strains is reported here, and several parameters of the specific and non-specific immune response in vaccinated trout were characterised. Vaccination with a dose of 10(7)cells/fish of the aroA mutant elicited significant protection against the Hooke and DK30 strains of A. salmonicida (relative percent survival RPS >60%). This cross-protection correlated moderately with the activation of the humoral and cellular specific immune responses, which show cross-reactivity against antigens shared by the two bacterial species, and a moderate increase in the lysozyme and antiprotease activities in the serum of vaccinated trout.

Topics: Aeromonas; Agglutination Tests; Analysis of Variance; Animals; Bacterial Vaccines; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Gram-Negative Bacterial Infections; Leukocytosis; Muramidase; Oncorhynchus mykiss; Protease Inhibitors; Vaccines, Attenuated

Immunisation of sea bream (Sparus aurata L.) broodstock with a novel vaccine mixture of Photobacterium damsela subsp. piscicida SK7 (Phdp) was performed during the period of egg development and the changes in specific and non-specific humoral immune parameters were measured. Total immunoglobulin level, specific antibody titre, anti-protease activity and lysozyme activity were significantly higher in immunised parents compared to the control. After spawning significantly higher anti-protease activity, lysozyme activity and total immunoglobulin level were detected in the eggs from immunised parents. Specific antibody titres against Phdp were only detected in the eggs from the immunised parents. The larvae from immunised parents also expressed significantly higher levels of specific and non-specific humoral immune parameters compared to the controls. A small amount of total immunoglobulin was detected in larvae decreasing gradually until day 8 post-hatching and then an increase was measured in larvae from immunised parents, whereas no immunoglobulin was detected at days 4, 6 and 8 in larvae from non-immunised parents. The specific antibody titre against Phdp was detected only in larvae from immunised broodstock until day 14 post-hatching. The higher humoral immune parameters in eggs and larvae from immunised parents in comparison to eggs and larvae from non-immunised parents, suggest transfer of maternal specific and non-specific immune factors.

Topics: Agglutination Tests; Analysis of Variance; Animals; Antibodies, Bacterial; Antibody Formation; Aquaculture; Blotting, Western; Electrophoresis; Enzyme-Linked Immunosorbent Assay; Fish Diseases; Gram-Negative Bacterial Infections; Immunization; Immunoglobulins; Larva; Muramidase; Ovum; Photobacterium; Sea Bream; Time Factors; Trypsin

The present work provides the first information concerning the immunostimulatory activity of trout recombinant interleukin-1 beta (rIL-1beta) in vivo. The predicted rainbow trout mature IL-1beta peptide was produced as a recombinant protein in Escherichia coli. Optimal migration of peritoneal leucocytes and rIL-1beta induced phagocytosis occurred following intraperitoneal injection of 1 microg of the recombinant protein. Moreover, systemic IL-1beta, COX-2 and lysozyme II gene expression was enhanced following >or=1 microg rIL-1beta administration. Finally, resistance to Aeromonas salmonicida, the causative agent of furunculosis, was augmented at early times (2 days) post-injection of 1 microg rIL-1beta.

Topics: Adjuvants, Immunologic; Aeromonas; Animals; Cyclooxygenase 2; Dose-Response Relationship, Drug; Gram-Negative Bacterial Infections; Immune System; Interleukin-1; Isoenzymes; Leukocyte Count; Leukocytes; Muramidase; Oncorhynchus mykiss; Phagocytosis; Prostaglandin-Endoperoxide Synthases

There has been increasing interest in the use of probiotics in aquaculture to control fish diseases (e.g. Douillet Langdon 1994; Gildberg, Mikkelsen, Sandaker & Ringo 1997; Kennedy, Tucker, Neidic, Vermeer, Cooper, Jarrell & Sennett 1998; DeSchrijver & Ollevier 2000; Robertson, O'Dowd, Burrells, Williams & Austin 2000), and a diverse range of Gram-positive (such as Carnobacterium inhibens) (Robertson et al. 2000) and Gram-negative bacteria, including Vibrio alginolyticus (Garriques & Arevalo 1995), have been evaluated at various times. However, the possible use of probiotics to control diseases of ornamental fish has been neglected. Consequently, following previous success with live (Irianto & Austin 2002) and inactivated probiotics in salmonids (Irianto & Austin, in press), research has focused on the usefulness of inactivated bacterial preparations for the control of atypical Aeromonas salmonicida infections in goldfish, Carassius auratus (L.). Goldfish, of average 40-50 mm in length, were obtained from a commercial farm in England, and maintained in aerated re-circulating dechlorinated fresh water at 17 degrees C. Their health status was examined immediately upon arrival in the aquaria and at 1-2-week periods thereafter (after Austin & Austin 1989).

Topics: Administration, Oral; Aeromonas hydrophila; Animals; Aquaculture; Erythrocyte Count; Fish Diseases; Formaldehyde; Goldfish; Gram-Negative Bacterial Infections; Leukocyte Count; Macrophages; Muramidase; Probiotics

The Arctic charr Salvelinus alpinus is an endangered fish species in Finland, and thus farming is carried out mainly for stocking purposes. Farmed charr are susceptible to infection with atypical Aeromonas salmonicida (aAS). Losses of valuable brood stock will severely reduce the genetic diversity of stocked charr. No commercial vaccines are available to prevent aAS infection, and vaccines against furunculosis (caused by typical A. salmonicida, tAS) do not protect the charr against aAS infection. The effects of a metabolizable oil-adjuvanted, bivalent vaccine (containing killed aAS and A. salmonicida salmonicida bacteria) on the immune system of 1 yr old hatchery-reared charr originating from Lake Inari in Northern Finland were examined. Fish vaccination in Finland generally takes place either from October to November or from February to April, when the water temperature is low (1 to 3degrees C). The water temperature starts to increase in mid-May. Therefore, we also investigated whether post-vaccination (p.v.) temperature had an influence on the immune system of this cold-water fish species. The fish were immunized intraperitoneally at 2.9 degrees C at the end of April. After 52 d, during which the water temperature increased from 2.9 to 10.0 degrees C, the charr were exposed to 1 of 3 test temperatures: 10.3, 14.1 or 18.1 degrees C. Prior to vaccination, and 49, 75 and 103 d p.v., several immune parameters were measured in both unvaccinated and vaccinated charr. Vaccination induced a significant anti-aAS-specific antibody response, and increased plasma lysozyme activity at all p.v. temperatures. The haemolytic activity of the complement system was unaffected either by vaccination or p.v. temperatures. There was a slight positive correlation between p.v. temperature and lysozyme activity of the charr. The significant increase in lysozyme activity took place in vaccinated charr in the first 49 d p.v. as water temperatures increased from 2.9 to 10 degrees C. Furthermore, the highest activity of lysozyme in the plasma was observed 49 d p.v. Our results indicate that a rise in water temperature above 10 degrees C does not significantly enhance the vaccination response of charr. This could be one reason why farmed Arctic charr, which are well adapted to a cold climate, are highly susceptible to aAS infection in the summer.

Topics: Aeromonas; Animals; Antibodies, Bacterial; Bacterial Vaccines; Breeding; Conservation of Natural Resources; Fish Diseases; Gram-Negative Bacterial Infections; Injections, Intraperitoneal; Muramidase; Seasons; Temperature; Time Factors; Trout; Vaccination

Recent research has identified endogenous cationic antimicrobial peptides as important factors in the innate immunity of many organisms, including fish. It is known that antimicrobial activity, as well as lysozyme activity, can be induced in coho salmon (Oncorhynchus kisutch) mucus after exposure of the fish to infectious agents. Since lysozyme alone does not have antimicrobial activity against Vibrio anguillarum and Aeromonas salmonicida, a four-step protein purification protocol was used to isolate and identify antibacterial fractions from bacterially challenged coho salmon mucus and blood. The purification consisted of extraction with hot acetic acid, extraction and concentration on a C(18) cartridge, gel filtration, and reverse-phase chromatography on a C(18) column. N-terminal amino acid sequence analyses revealed that both the blood and the mucus antimicrobial fractions demonstrated identity with the N terminus of trout H1 histone. Mass spectroscopic analysis indicated the presence of the entire histone, as well as fragments thereof, including a 26-amino-acid N-terminal segment. These fractions inhibited the growth of antibiotic-supersuscptible Salmonella enterica serovar Typhimurium, as well as A. salmonicida and V. anguillarum. Synthetic peptides identical to the N-terminally acetylated or C-terminally amidated 26-amino-acid fragment were inactive in antimicrobial assays, but they potentiated the antimicrobial activities of the flounder peptide pleurocidin, lysozyme, and crude lysozyme-containing extracts from coho salmon. The peptides bound specifically to anionic lipid monolayers. However, synergy with pleurocidin did not appear to occur at the cell membrane level. The synergistic activities of inducible histone peptides indicate that they play an important role in the first line of salmon defenses against infectious pathogens and that while some histone fragments may have direct antimicrobial effects, others improve existing defenses.

Topics: Aeromonas; Animals; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Fish Diseases; Fish Proteins; Flounder; Gram-Negative Bacterial Infections; Histones; Mucus; Muramidase; Oncorhynchus kisutch; Peptides; Proteins; Vibrio; Vibrio Infections

Utilization of natural immunostimulants in fish culture offers a wide range of attractive methods for inducing and building protection against diseases. Lysozyme is an enzyme with bacteriolytic properties and is ubiquitous in its distribution among living organisms. This enzyme has antiviral, antibacterial and anti-inflammatory properties. In nature, lysozyme is found as a monomer. Lysozyme dimer is significantly less toxic than its monomer, and its high biological activity has been ascertained in cases of both viral and bacterial infections. In our study, we examined the influence of dimerized lysozyme (KLP-602) on the nonspecific cellular and humoral defense mechanisms and protection against furunculosis in rainbow trout (Oncorhynchus mykiss). We have analyzed the immunomodulatory effects of KLP-602 after experimental infection by Aeromonas salmonicida. Application of dimerized lysozyme (KLP-602) by injection stimulated the cellular and humoral defense mechanisms and provided protection against furunculosis. By contrast, mortality rate was reduced to 45% (one injection) and 25% (three injections) using 10 or 100 microg/kg KLP-602. Mortality in the untreated control group was 85%.

Topics: Adjuvants, Immunologic; Aeromonas; Animals; Cytotoxicity, Immunologic; Dimerization; Fish Diseases; Furunculosis; Gram-Negative Bacterial Infections; Immunity, Cellular; Immunoglobulins; Muramidase; Oncorhynchus mykiss; Phagocytes; Respiratory Burst; Time Factors

The protective effect of lysozyme-galactomannan or lysozyme-palmitic acid conjugates orally administered to carp, Cyprinus carpio L. was investigated using a virulent strain of Gram-negative Edwardsiella tarda isolated from an infected fish. Lysozyme-galactomannan conjugate was prepared through controlled Maillard reaction. Lysozyme-palmitic acid conjugate was prepared through base-catalyzed ester exchange using N-hydroxysuccinimide ester of palmitic acid. The conjugates provided substantial protection to carp infected with a Gram-negative bacteria fish pathogen E. tarda NG 8104. Lytic activities of lysozyme conjugates with galactomannan and palmitic acid were about 80 and 71% of native lysozyme using Micrococcus lysodeikticus as a substrate. Feeding with lysozyme conjugates, for 8 days, significantly enhanced fish protection against E. tarda infection. The survival rate was 30% for lysozyme-galactomannan conjugate treated fish and 20% for lysozyme-palmitic acid conjugate treated fish after 6 days cultivation while all control fish died within 3 days. On the other hand, a recovery rate of 40% after 6 days was observed in the fish group that were fed lysozyme-palmitic acid conjugate 3 and 2 h before and after E. tarda challenge, respectively, and for 6 consecutive days. The results of this work show the possibility of utilizing lysozyme conjugates with galactomannan or palmitic acid as a therapeutic for infection in fish.

Topics: Animals; Anti-Bacterial Agents; Fish Diseases; Galactose; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Maillard Reaction; Mannans; Microbial Sensitivity Tests; Muramidase; Palmitic Acid; Time Factors; Virulence

The authors have compared the antimicrobial resistance patterns and plasmid profiles of Gram-negative isolates in an intensive care unit over a 7-month period in order to identify epidemiologically related isolates. Bacterial plasmids were found to be valuable markers for the comparison of strains of nosocomial Gram-negative bacilli. Thirty-nine mechanically ventilated patients in an ICU were included. From bronchoaspiratus, the authors isolated 58 strains of Gram-negative bacilli (24 Ps. aeruginosa and 34 Enterobacteria). Common plasmids were found in most Enterobacteria. The interspecies plasmid exchange suggests that interstate spread of these strains may have occurred. Twenty-six Enterobacteria carried plasmids, 11 of which proved transmissible. The R-factors were transferred to other genera that were isolated in the hospital, thereby adding to the pool of multiresistant nosocomial isolates. Larger plasmids transferred ampicillin and carbenicillin resistance, while gentamycin and cephalotin resistance was carried by smaller plasmids. Only 4 Ps. aeruginosa carried plasmids, one of which was transmissible. Pseudomonas plasmid DNA is extracted with difficulty by the simple lysis method, due to the roughness of the colonies. All Pseudomonas isolates belonged to the same biotype which can be regarded as an epidemiological marker. Therefore, plasmid profiling is a useful tool for epidemiological surveillance of Enterobacteria and is a good method for determining the relatedness of isolates in a nosocomial environment.

Topics: Biomarkers; Cross Infection; DNA Restriction Enzymes; Electrophoresis, Polyacrylamide Gel; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Humans; Intensive Care Units; Microbial Sensitivity Tests; Middle Aged; Muramidase; Plasmids; Pyocins; R Factors; Serotyping