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1.
Int J Biol Macromol ; 257(Pt 1): 128367, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38029897

ABSTRACT

A multifunctional alginate/PDRN hydrogel system by ionic crosslinking and the Schiff base reaction between oxidized alginate (OA) and PDRN was developed in the present study. Biocompatibility assessment of the PDRN-loaded OA hydrogels showed a significant enhancement in cell viability in human dermal fibroblast (HDF) cells. In addition, hydrogels showed migratory, anti-inflammatory, intracellular reactive oxygen species scavenging, and anti-apoptotic activities. In vivo studies using a streptozotocin-induced diabetic Wister rat model indicated that OA-4PDRN had the highest percentage of wound closure (96.1 ± 2.6 %) at day 14 compared to the control (79.0 ± 2.3 %) group. This was accompanied by up-regulation of vascular endothelial growth factor (VEGF), interleukin-10 (IL-10), and transforming growth factor-beta (TGF-ß) accompanied by down-regulation of pro-inflammatory markers (IL-6, IL-1ß). Following histopathological observations, PDRN-loaded OA hydrogel ensured tissue safety and induced wound healing with granular tissue formation, collagen deposition, re-epithelialization, and regeneration of blood vessels and hair follicles. The downregulation of inflammatory cytokines (CD68) and expression of angiogenesis-related cytokines (CD31) in wound sites revealed the suppression of inflammation and increased angiogenesis, ensuring skin tissue regeneration in diabetic wound healing. In conclusion, the findings suggest that PDRN-loaded OA hydrogel has enormous therapeutic potential as a diabetic wound dressing.


Subject(s)
Diabetes Mellitus , Hydrogels , Rats , Humans , Animals , Hydrogels/pharmacology , Vascular Endothelial Growth Factor A/metabolism , Polydeoxyribonucleotides/pharmacology , Alginates , Rats, Wistar , Wound Healing , Cytokines
2.
Int J Biol Macromol ; 253(Pt 3): 126729, 2023 Dec 31.
Article in English | MEDLINE | ID: mdl-37689303

ABSTRACT

Polydeoxyribonucleotide (PDRN) is an accelerated diabetic wound healing therapy with promising abilities to promote cell growth, angiogenesis, collagen synthesis, and reduce inflammation where its sustainable delivery and release behavior is critical to ensure effective wound healing properties. Therefore, a nanopolyplex was developed here, by encapsulating PDRN with chitosan to affirm its delivery systematically. The physicochemical characterization revealed its successful encapsulation which facilitates the gradual release of PDRN. In vitro studies of the polyplex demonstrated no cytotoxicity and enhanced cell proliferation and migration properties with high antimicrobial activities. In vivo, wound healing studies in Wistar rats dorsal skin defect model induced with diabetes mellitus affirm the highest wound healing activity and wound closure rate by chitosan/PDRN polyplex treatment. Considerably high histopathological changes such as epithelialization, collagen deposition, blood vessels, and hair follicle formation were observed under the polyplex treatment. The immunohistochemical analysis for platelet endothelial cell adhesion molecule (CD31) and cluster of differentiation (CD68) revealed the ability of polyplex to increase CD31 expression and decrease CD68 expression thereby promoting the wound healing process. Collectively, these results suggest that significantly accelerated, high-quality wound healing effects could be obtained by the developed chitosan/PDRN polyplex and thus it could be introduced as a potential therapeutic product for diabetic wound healing.


Subject(s)
Chitosan , Diabetes Mellitus , Rats , Animals , Chitosan/pharmacology , Polydeoxyribonucleotides/pharmacology , Polydeoxyribonucleotides/therapeutic use , Rats, Wistar , Wound Healing , Collagen/pharmacology , Diabetes Mellitus/drug therapy
3.
Int J Mol Sci ; 23(21)2022 Nov 04.
Article in English | MEDLINE | ID: mdl-36362312

ABSTRACT

This study aimed to develop a corneal epithelial injury model in zebrafish (Danio rerio) and investigate the effectiveness of polydeoxyribonucleotide (PDRN) treatment on in vivo corneal epithelial regeneration and wound healing. Chemical injury to zebrafish cornea was produced by placing a small cotton swab containing 3% acetic acid solution. PDRN treatment was performed by immersing corneal-injured zebrafish in water containing PDRN (2 mg/mL) for 10 min at 0, 24, 48, and 72 h post-injury (hpi). The level of corneal healing was evaluated by fluorescein staining, histological examination, transcriptional profiling, and immunoblotting techniques. Fluorescein staining results demonstrate that PDRN treatment significantly (p < 0.05) reduced the wounded area of the zebrafish eye at 48 and 72 hpi, suggesting that PDRN may accelerate the corneal re-epithelialization. Histopathological evaluation revealed that injured corneal epithelial cells were re-organized at 72 hpi upon PDRN treatment with increased goblet cell density and size. Moreover, transcriptional analysis results demonstrate that PDRN treatment induced the mRNA expression of adora2ab (6.3-fold), pax6a (7.8-fold), pax6b (29.3-fold), klf4 (7.3-fold), and muc2.1 (5.0-fold) after the first treatment. Besides, tnf-α (2.0-fold) and heat-shock proteins (hsp70; 2.8-fold and hsp90ab1; 1.6-fold) have modulated the gene expression following the PDRN treatment. Immunoblotting results convincingly confirmed the modulation of Mmp-9, Hsp70, and Tnf-α expression levels upon PDRN treatment. Overall, our corneal injury model in zebrafish allows for understanding the morphological and molecular events of corneal epithelial healing, and ophthalmic responses for PDRN treatment following acid injury in zebrafish.


Subject(s)
Corneal Injuries , Polydeoxyribonucleotides , Animals , Polydeoxyribonucleotides/pharmacology , Polydeoxyribonucleotides/therapeutic use , Zebrafish , Tumor Necrosis Factor-alpha/pharmacology , Corneal Injuries/drug therapy , Corneal Injuries/metabolism , Wound Healing , Cornea/metabolism , Fluoresceins/pharmacology
4.
Arch Virol ; 167(4): 1225-1230, 2022 Apr.
Article in English | MEDLINE | ID: mdl-35296935

ABSTRACT

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


Subject(s)
Aeromonas , Genome, Viral , Myoviridae/genetics , Phylogeny , Sequence Analysis
5.
Int J Biol Macromol ; 193(Pt B): 1823-1834, 2021 Dec 15.
Article in English | MEDLINE | ID: mdl-34785202

ABSTRACT

Development of novel wound dressing materials having the ability to prevent bacterial infections and capable of accelerating the tissue regeneration process is utmost important, since the wounds in patients can cause severe health issues. In the present work, we synthesized novel N-succinyl chitosan nanoparticles (N-SuC NPs) films and tested their antimicrobial, cytotoxicity, and in vitro and in vivo wound healing activity. N-SuC NPs were synthesized by ionic gelation method, and subsequently N-SuC NPs films were prepared by solution casting method using synthesized N-SuC NPs. The prepared N-SuC NPs films showed significant antimicrobial activity against Escherichia coli and Staphylococcus aureus with a minimum inhibitory concentration of 6 mg/mL and <8 mg/mL, respectively. The biocompatibility and the in vitro wound healing activity of N-SuC NPs films were analyzed using human dermal fibroblast (HDF) cells. In vivo cutaneous wound healing of the N-SuC NPs film was investigated using the Wister rat model, and the studies showed that the N-SuC NPs film significantly accelerated the wound healing process by inducing more blood vessels formation and tissue granulation. The experimental results showed that synthesized N-SuC NPs film had excellent antimicrobial, cytotoxicity and wound healing activity, indicating that it could be used in biomedical applications.


Subject(s)
Chitosan , Escherichia coli/growth & development , Membranes, Artificial , Nanoparticles , Staphylococcus aureus/growth & development , Wound Healing/drug effects , Animals , Cell Line , Chitosan/chemistry , Chitosan/pharmacology , Humans , Nanoparticles/chemistry , Nanoparticles/therapeutic use , Rats , Rats, Wistar
6.
Fish Shellfish Immunol ; 110: 23-34, 2021 Mar.
Article in English | MEDLINE | ID: mdl-33378697

ABSTRACT

Streptococcus parauberis is a pathogenic gram-positive bacterium that causes streptococcosis infection in fish. Since S. parauberis is becoming resistant to multiple antibiotics, the development of alternatives, such as antimicrobial peptides, has gained great attention. Octominin, derived from the defense protein of Octopus minor, showed a significant antimicrobial activity against multidrug resistance S. parauberis, with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 50 and 100 µg/mL, respectively. Furthermore, time-kill kinetics, agar diffusion, and bacterial viability assays confirmed the concentration-dependent antibacterial activity of Octominin against S. parauberis. Field emission scanning electron microscopy analysis showed morphological and ultra-structural changes in S. parauberis upon Octominin treatment. Moreover, Octominin treatment demonstrated changes in membrane permeability, induced reactive oxygen species (ROS), and its binding ability to genomic DNA, suggesting its strong bactericidal activity with multiple modes of action. We confirmed the inhibition of biofilm formation and the eradication of existing biofilms in a concentration-dependent manner. Additionally, Octominin on S. parauberis at transcriptional level exhibited downregulation of membrane formation (pgsA and cds1), DNA repairing (recF), biofilm formation (pgaB and epsF) genes, while upregulation of ROS detoxification (sodA) and DNA protecting (ahpF) related genes. An in vivo study confirmed a significantly (P < 0.05) higher relative percentage survival in Octominin-treated larval zebrafish exposed to S. parauberis (93.3%) compared to the control group (20.0%). Collectively, our results confirm that Octominin could be a potential antibacterial and anti-biofilm agent against S. parauberis.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Biofilms/drug effects , Microbial Viability/drug effects , Peptide Fragments/pharmacology , Streptococcus/drug effects , Animals , Drug Resistance, Multiple, Bacterial , Fish Diseases/prevention & control , Microbial Sensitivity Tests/veterinary , Microscopy, Electron, Scanning , Streptococcal Infections/prevention & control , Streptococcal Infections/veterinary , Streptococcus/physiology , Streptococcus/ultrastructure
7.
Int J Biol Macromol ; 165(Pt A): 63-70, 2020 Dec 15.
Article in English | MEDLINE | ID: mdl-32971172

ABSTRACT

Herein, we have successfully synthesized a novel N-Succinyl chitosan/gold nanocomposite (N-SuC/Au NC) using N-SuC and gold(III) chloride, and investigated the biocompatibility and antifungal activity. The synthesized N-SuC/Au NC was characterized by UV-visible spectroscopy, X-ray diffraction, field emission scanning electron microscope, and inductively coupled plasma atomic emission spectroscopy. The N-SuC/Au NC exhibited a strong inhibition effect towards pathogenic Candida albicans. Morphological analysis revealed the destruction of C. albicans cell membrane due to N-SuC/Au NC treatment. The in vitro and in vivo toxicity of N-SuC/Au NC was analyzed with HEK293T mammalian cells and zebrafish larvae, respectively. The synthesized N-SuC/Au NC demonstrated no cytotoxicity towards HEK293T cells up to 1200 µg/mL concentration. The survival rate of the zebrafish larvae at 120 hpf, was found as 100% up to 1200 µg/mL of N-SuC/Au NC exposure. The in vivo studies further confirmed the inhibitory effects of N-SuC/Au NC on the formation of C. albicans hyphae in infected zebrafish muscle tissue.


Subject(s)
Antifungal Agents/chemistry , Candida albicans/drug effects , Chitosan/chemistry , Nanocomposites/chemistry , Animals , Antifungal Agents/pharmacology , Candida albicans/pathogenicity , Chitosan/pharmacology , Drug Carriers/chemistry , Drug Carriers/pharmacology , Gold/chemistry , HEK293 Cells , Humans , Silver/chemistry , X-Ray Diffraction
8.
Braz J Microbiol ; 51(4): 2153-2162, 2020 Dec.
Article in English | MEDLINE | ID: mdl-32651888

ABSTRACT

This study proposed that phage-enriched artemia could be a useful tool for transferring phage into the cultured fish (larvae or adult) as a feed, and introduce mode of phage administration and its safety in concern of tissue adaptation for efficient phage therapy in aquatic animals. First, whether Edwardsiella tarda phage (ETP-1) could attach or ingest by the artemia and optimum time period for the ETP-1 enrichment with artemia were investigated. ETP-1 dispersion, abundance and persistency, and zebrafish immune transcriptional responses and histopathology were evaluated after feeding the fish with ETP-1-enriched artemia. Hatched artemia nauplii (36 h) were enriched with 1.90 × 1011 PFUmL-1 of ETP-1, and maintained at 25 °C. The highest enrichment level was obtained after 4 h (3.00 × 109 PFUmL-1), and artemia were alive and active similar to control for 8 h. ETP-1 disseminated dose dependently to all the tissues rapidly (12 h). However, when feeding discontinued, it drastically decreased at day 3 with high abundance and persistency in the spleen (1.02 × 103) followed by the kidney (4.00 × 101) and the gut (1 × 101 PFUmL-1) for highest ETP-1-enriched artemia dose. In contrast, during continuous delivery of ETP-1-enriched artemia, ETP-1 detected in all the tissues (at day 10: gut; 1.90 × 107, kidney; 3.33 × 106, spleen; 5.52 × 105, liver; 6.20 × 104 PFUmL-1mg-1 tissues). Though the phage abundance varied, results indicated that oral fed ETP-1-enriched artemia disperse to the neighboring organs, even the absence of host as phage carrier. Non-significant differences of immune transcriptional and histopathology analysis between ETP-1-enriched artemia fed and controls suggest that no adverse apparent immune stimulation in host occurred, and use of ETP-1 at 1011 PFUmL-1 was safe. With further supportive studies, live artemia-mediated phage delivery method could be used as a promising tool during phage therapy against pathogenic bacteria to control aquatic diseases.


Subject(s)
Animal Feed/virology , Artemia/virology , Edwardsiella tarda/virology , Phage Therapy/methods , Animal Feed/analysis , Animals , Aquaculture/methods , Fish Diseases/therapy , Microspheres , Transcriptome , Zebrafish/immunology , Zebrafish/virology
9.
Mar Drugs ; 18(3)2020 Mar 21.
Article in English | MEDLINE | ID: mdl-32245246

ABSTRACT

This study evaluated the modulation of gut microbiota, immune responses, and gut morphometry in C57BL/6 mice, upon oral administration of S. maxima-derived modified pectin (SmP, 7.5 mg/mL) and pectin nanoparticles (SmPNPs; 7.5 mg/mL). Metagenomics analysis was conducted using fecal samples, and mice duodenum and jejunum were used for analyzing the immune response and gut morphometry, respectively. The results of metagenomics analysis revealed that the abundance of Bacteroidetes in the gut increased in response to both modified SmP and SmPNPs (75%) as compared with that in the control group (66%), while that of Firmicutes decreased in (20%) as compared with that in the control group (30%). The mRNA levels of mucin, antimicrobial peptide, and antiviral and gut permeability-related genes in the duodenum were significantly (p < 0.05) upregulated (> 2-fold) upon modified SmP and SmPNPs feeding. Protein level of intestinal alkaline phosphatase was increased (1.9-fold) in the duodenum of modified SmPNPs feeding, evidenced by significantly increased goblet cell density (0.5 ± 0.03 cells/1000 µm2) and villi height (352 ± 10 µm). Our results suggest that both modified SmP and SmPNPs have the potential to modulate gut microbial community, enhance the expression of immune related genes, and improve gut morphology.


Subject(s)
Gastrointestinal Microbiome/drug effects , Microalgae/chemistry , Nanoparticles/administration & dosage , Pectins/administration & dosage , Prebiotics/administration & dosage , Spirulina/chemistry , Animals , Antimicrobial Cationic Peptides/analysis , Antimicrobial Cationic Peptides/metabolism , Bacteroidetes/genetics , Bacteroidetes/isolation & purification , Feces/microbiology , Gastrointestinal Microbiome/genetics , Immunity, Innate/drug effects , Immunity, Mucosal/drug effects , Intestinal Mucosa/drug effects , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Male , Metagenomics , Mice , Models, Animal , Mucins/analysis , Mucins/metabolism , Pectins/isolation & purification
10.
Mar Drugs ; 18(1)2020 Jan 15.
Article in English | MEDLINE | ID: mdl-31952292

ABSTRACT

: The rapid emergence of multidrug-resistant pathogens makes an urgent need for discovering novel antimicrobial agents as alternatives to conventional antibiotics. Towards this end, we designed and synthesized a synthetic peptide of 23 amino acids (AAs) (1GWLIRGAIHAGKAIHGLIHRRRH23) from a defense protein 3 cDNA sequence of Octopus minor. The sequence of the peptide, which was named Octominin, had characteristic features of known antimicrobial peptides (AMPs) such as a positive charge (+5), high hydrophobic residue ratio (43%), and 1.86 kcal/mol of Boman index. Octominin was predicted to have an alpha-helix secondary structure. The synthesized Octominin was 2625.2 Da with 92.5% purity. The peptide showed a minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 50 and 200 µg/mL, respectively, against Candida albicans. Field emission scanning electron microscopy observation confirmed that Octominin caused ultrastructural cell wall deformities in C. albicans. In addition, propidium iodide penetrated the Octominin-treated C. albicans cells, further demonstrating loss of cell membrane integrity that caused cell death at both MIC and MFC. Octominin treatment increased the production of intracellular reactive oxygen species and decreased cell viability in a concentration dependent manner. Cytotoxicity assays revealed no significant influence of Octominin on the viability of human embryonic kidney 293T cell line, with over 95% live cells in the Octominin-treated group observed up to 100 µg/mL. Moreover, we confirmed the antifungal action of Octominin in vivo using a zebrafish experimental infection model. Overall, our results demonstrate the Octominin is a lead compound for further studies, which exerts its effects by inducing cell wall damage, causing loss of cell membrane integrity, and elevating oxidative stress.


Subject(s)
Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Candida albicans/drug effects , Octopodiformes/chemistry , Animals , Cell Membrane/drug effects , Cell Survival/drug effects , Cell Wall/drug effects , Cells, Cultured , HEK293 Cells , Humans , Microbial Sensitivity Tests/methods , Oxidative Stress/drug effects , Protein Conformation, alpha-Helical , Reactive Oxygen Species/metabolism , Zebrafish
11.
Biologicals ; 63: 14-23, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31889622

ABSTRACT

Edwardsiella tarda phage (ETP-1) was isolated from marine fish farm water to characterize its effect against pathogenic multidrug-resistant E. tarda. According to transmission electron microscopy results, ETP-1 is classified as a member of the family Podoviridae. ETP-1 showed MOI dependent E. tarda growth inhibition, a latent period of 60 min, and burst size of 100 PFU per infected cells. In host range tests, five out of eight E. tarda strains were sensitive to ETP-1 which had efficiency of plating index in the range 1-1.28. ETP-1 was stable over a broad range of pH and temperature. The size of the ETP-1 genome was predicted to be approximately 40 kb. Zebrafish exposed to ETP-1 showed no adverse gene responses to the inflammatory mediator cytokines, il1-ß, tnf-α, il-6, and il-10, the chemokine, cxcl-8a, and reactive oxygen species, sod-1. When zebrafish were bath exposed to ETP-1 for 12 days and simultaneously challenged with E. tarda (1.08 × 105 CFU fish-1), the survival rate was higher in phage exposed fish (68%) compared to that of the control (18%) until 4 days post challenge. Our results suggest that ETP-1 can be used as a potential bio-therapeutic candidate to control multi-drug resistant E. tarda infection in aquaculture.


Subject(s)
Drug Resistance, Multiple, Bacterial , Edwardsiella tarda , Enterobacteriaceae Infections/therapy , Fish Diseases , Phage Therapy , Podoviridae , Animals , Edwardsiella tarda/pathogenicity , Edwardsiella tarda/virology , Fish Diseases/microbiology , Fish Diseases/therapy , Zebrafish
12.
Braz J Microbiol ; 51(1): 409-416, 2020 Mar.
Article in English | MEDLINE | ID: mdl-31691176

ABSTRACT

To develop an alternative bio-control measure for multi-drug resistant pathogenic Aeromonas hydrophila, which causes motile Aeromonas septicemia in fish, novel virulent phage (AHP-1) was isolated from carp tissues. Morphological analysis by transmission electron microscopy revealed that AHP-1 belongs to Myoviridae family. AHP-1 displayed 81% of moderate adsorption by 25 min, and latent period of 40 min with burst size of 97 PFU mL-1 at an optimal multiplicity of infection (MOI) 0.1. AHP-1 was stable over a broad range of pH (4-11), temperature (4-50 °C), and salinity (0.1-3.5%). Both time and MOI dependent in vitro A. hydrophila growth inhibition was observed with AHP-1. AHP-1 (10 MOI) showed higher growth inhibition against A. hydrophila than chloramphenicol (5 µg mL-1), and combined treatment was more promising than individuals. Immune gene expression analysis of zebrafish upon continuous bath exposure to AHP-1 resulted significantly higher (il-6 and sod-1) or slight induction (tnf-α, il1-ß, il-10, and cxcl-8a) than controls at beginning of the phage exposure, but those lowered to basal level by day 12 post-phage exposure. It suggests no adverse immune responses have occurred for the AHP-1 dose that used, and have potential for the phage therapy. Further detailed in vivo studies are needed to confirm the protective efficacy of newly isolated AHP-1 against A. hydrophila infection.


Subject(s)
Aeromonas hydrophila , Fish Diseases/microbiology , Myoviridae/isolation & purification , Zebrafish/immunology , Aeromonas hydrophila/drug effects , Aeromonas hydrophila/virology , Animals , Bacteriophages/immunology , Bacteriophages/isolation & purification , Bacteriophages/ultrastructure , Biological Control Agents , Carps/virology , Chloramphenicol/pharmacology , Fish Diseases/therapy , Fishes , Immunity, Cellular , Myoviridae/immunology , Myoviridae/ultrastructure , Zebrafish/microbiology , Zebrafish/virology
13.
Fish Shellfish Immunol ; 94: 558-565, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31546036

ABSTRACT

In this study, we demonstrate the enhanced disease resistance and positive immunomodulation of novel pectin isolated from Spirulina maxima (SmP) in zebrafish model. Zebrafish larvae exposed to SmP had significantly (p < 0.05) higher cumulative percent survival (CPS) at 25 (44.0%) and 50 µg/mL (67.0%) against Edwardsiella piscicida compared to the control. However, upon Aeromonas hydrophila challenge, SmP exposed larvae at 50 µg/mL had slightly higher CPS (33.3%) compared to control group (26.7%). SmP supplemented zebrafish exhibited the higher CPS against E. piscicida (93.3%) and A. hydrophila (60.0%) during the early stage of post-infection (<18 hpi). qRT-PCR results demonstrated that exposing (larvae) and feeding (adults) of SmP, drive the modulation of a wide array of immune response genes. In SmP exposed larvae, up-regulation of the antimicrobial enzyme (lyz: 3.5-fold), mucin (muc5.1: 2.84, muc5.2: 2.11 and muc5.3: 2.40-fold), pro-inflammatory cytokines (il1ß: 1.79-fold) and anti-oxidants (cat: 2.87 and sod1: 1.82-fold) were identified. In SmP fed adult zebrafish (gut) showed >2-fold induced pro-inflammatory cytokine (il1ß) and chemokines (cxcl18b, ccl34a.4 and ccl34b.4). Overall results confirmed the positive modulation of innate immune responses in larval stage and it could be the main reason for developing disease resistance against E. piscicida and A. hydrophila. Thus, non-toxic, natural and biodegradable SmP could be considered as the potential immunomodulatory agent for sustainable aquaculture.


Subject(s)
Cyanobacteria/chemistry , Disease Resistance/drug effects , Fish Diseases/immunology , Immunity, Innate/drug effects , Pectins/metabolism , Zebrafish/metabolism , Aeromonas hydrophila/physiology , Animal Feed/analysis , Animals , Bacterial Proteins/administration & dosage , Bacterial Proteins/metabolism , Diet/veterinary , Dietary Proteins/administration & dosage , Dietary Proteins/metabolism , Dietary Supplements/analysis , Edwardsiella/physiology , Enterobacteriaceae Infections/immunology , Enterobacteriaceae Infections/veterinary , Gram-Negative Bacterial Infections/immunology , Gram-Negative Bacterial Infections/veterinary , Pectins/administration & dosage
14.
Mycopathologia ; 184(5): 559-572, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31473909

ABSTRACT

BACKGROUND: Candida albicans is an opportunistic fungal pathogen which causes systemic infections in human. In this study, C. albicans infection model was developed in zebrafish to understand the host-pathogen interactions for straightforward anticandidal drug screening. METHODS: To develop the infection, 1 × 106 cells of C. albicans suspended in phosphate-buffered saline were deposited in zebrafish dorsal muscle by manually operated syringe. The infection progression was externally assessed by a scale of wound-healing events, based on visible changes of yeast deposited in the muscle tissues. Chemotherapy was carried out with known antifungal drugs (fluconazole, nystatin, and amphotericin B) and a potential antifungal agent, chitosan silver nanocomposites (CAgNC), after the infection as direct exposure in the water. Histopathological analysis was performed to identify the pathogen virulence and the host-pathogen interaction during the infection. RESULTS: The light microscopic observations and histopathological analysis revealed the yeast-hyphae transition at the site of infection (at 72 hpi) and progression of the infection in the host tissues. The larval survival rate under fluconazole (up to 80 µg mL-1) and nystatin (up to 20 µg mL-1) was > 90% and for CAgNC it was 40% at 36 h post-exposure (hpe). The infection progression was suppressed with the fungicidal treatments. Among inflammatory genes, il-1ß has been highly upregulated (14.68-fold) at 24 h post infection (hpi). Both il-1ß and tnf-α were moderately upregulated in infected fish gills at 72 hpi. Among the C. albicans antioxidant genes, cat1 and sod2 have been upregulated during the infection, and relative expression folds were increased from low to moderate levels with the time. DISCUSSION: We demonstrate the approach for the development of artificial infection model of zebrafish with C. albicans. By this mini vertebrate zebrafish model, researchers will be able to study novel anticandidal compounds in vivo with respect to the host, pathogen, and their interactions.


Subject(s)
Antifungal Agents/isolation & purification , Antifungal Agents/pharmacology , Candida albicans/drug effects , Candidiasis/microbiology , Disease Models, Animal , Drug Evaluation, Preclinical/methods , Animals , Candida albicans/pathogenicity , Candidiasis/pathology , Histocytochemistry , Host-Pathogen Interactions , Microscopy , Zebrafish
15.
Indian J Microbiol ; 59(2): 161-170, 2019 Jun.
Article in English | MEDLINE | ID: mdl-31031430

ABSTRACT

In this study, Aeromonas salmonicida subsp. salmonicida was isolated, identified by 16S RNA sequencing and its potential lytic phage (ASP-1) was isolated and characterized. The bacterium was positive for virulence genes (ascV, fla, ahyB, gcaT, lip, alt and act) and phenotypic parameters (haemolysis, slime production, lipase activity, DNase test, gelatinase activity and protease activity) were tested. The bacterium was resistant to 27%, intermediate resistant to 14% and susceptible to 59% of tested common antibiotics. Transmission electron microscopy analysis revealed that lytic ASP-1 belongs to the Myoviridae family. The isolated phage was more specific against A. salmonicida subsp. salmonicida (efficiency of plating index = 1), but also had infectivity to A. hydrophila lab strain 1. The bacteriolytic effect of ASP-1 was tested at early exponential phase culture of A. salmonicida subsp. salmonicida, and bacteria growth was apparently decreased with time and MOI dependent manner. One-step growth of ASP-1 showed approximately 30 min of latent period, 16 PFU/infected cells of burst size and 40 min of rise period. The adsorption rate was determined as 3.61 × 108 PFU mL-1 min-1 for 3 min, and rate decreased with time. The ASP-1 genome size was estimated to be approximately 55-60 kD. The phage was stable over wide-range of temperatures, pH and salinity, thus could withstand at severe environmental conditions, indicating that ASP-1 has a potential to develop as an alternative antibiotic to use in ornamental and aquaculture industry.

16.
Fish Shellfish Immunol ; 84: 1030-1040, 2019 Jan.
Article in English | MEDLINE | ID: mdl-30359749

ABSTRACT

Proteins with dithiol-disulfide oxidoreductase catalytic domain are well known for their capacity in the cellular redox homeostasis. In this study, we characterized the zebrafish thioredoxin domain containing 12 (Zftxndc12) gene, analyzed the transcriptional responses and studied the functional properties of its recombinant protein. Full-length cDNA of Zftxndc12 consists 519 bp coding region encoding 172 amino acids (AA) including the signal peptide. Highly consensus active motif (65WCGAC69) and probable ER retrieval motif (169GDEL172) were identified. Ubiquitous expression of Zftxndc12 mRNA was observed from one cell to juvenile stage as well as different organs of adult zebrafish. Moreover, whole mount in situ hybridization (WISH) results showed a higher expression of Zftxndc12 in primordial gills, central nerves system and eye. The tissue specific expression analysis (by qRT-PCR) also showed the highest expression in gills followed by brain in adult zebrafish. In larvae, up-regulated Zftxndc12 mRNA expression upon exposure to H2O2,Edwardsiella tarda and Saprolegnia parasitica suggests that it may involve in both stress and immune responses. Moreover, transcriptional expression of Zftxndc12 was up-regulated upon Streptococcus iniae challenge in gills of adult zebrafish. The recombinant ZfTxndc12 (rZfTxndc12) was overexpressed, purified and tested for its biological activities. Results revealed that rZfTxndc12 is able to reduce the DNA damage and detoxify the H2O2 toxicity in concentration dependent manner. Overall results suggest that Zftxndc12 is important antioxidant and immune functional member of the host defense system in zebrafish.


Subject(s)
Fish Diseases/immunology , Gene Expression Regulation/immunology , Immunity, Innate/genetics , Zebrafish/genetics , Zebrafish/immunology , Amino Acid Sequence , Animals , Base Sequence , Edwardsiella tarda/physiology , Enterobacteriaceae Infections/immunology , Enterobacteriaceae Infections/veterinary , Gene Expression Profiling/veterinary , Infections/immunology , Infections/veterinary , Phylogeny , Saprolegnia/physiology , Sequence Alignment/veterinary , Streptococcal Infections/immunology , Streptococcal Infections/veterinary , Streptococcus iniae/physiology
17.
Fish Shellfish Immunol ; 80: 573-581, 2018 Sep.
Article in English | MEDLINE | ID: mdl-29964197

ABSTRACT

Fish can be potentially co-infected by two or more bacterial strains, which can make synergistic influence on the virulence of infection. In this study, two opportunistic and multidrug resistant Aeromonas strains were isolated from wounds of morbid zebrafish with typical deep skin lesions similar to Motile Aeromonas Septicemia. Isolates were genetically identified as A. hydrophila and A. veronii by 16 S rRNA sequencing and phylogenetic analysis. Both isolates were positive for virulent genes (aerA, lip, ser, exu gcaT) and selected phenotypic tests (DNase, protease, gelatinase, lipase, biofilm production and ß-haemolysis). A. hydrophila and A. veronii had strong antibiotic resistance against ampicillin, tetracycline, nalidixic acid, kanamycin, erythromycin, clindamycin and trimethoprim-sulfamethoxazole. Histopathological studies revealed that co-infection causes severe necrosis and hypertrophy in the muscles, kidney and liver of zebrafish. Naturally co-infected zebrafish showed highly induced tnf-α, il-1ß, il-6, il-12, ifn, ifn-γ, cxcl18 b and ccl34a.4 at transcription level compared to healthy fish, suggesting virulence factors may activate immune and inflammatory responses of zebrafish. Experimentally infected zebrafish showed significantly higher mortality under co-infection with A. hydrohila and A. veronii (87%), followed by individual challenge of A. hydrophila (72%) or A. veronii (67%) suggesting that virulence of A. hydrophila have greater pathogenicity than A. veronii during co-infection.


Subject(s)
Aeromonas , Coinfection , Fish Diseases , Gram-Negative Bacterial Infections , Zebrafish , Aeromonas/drug effects , Aeromonas/genetics , Aeromonas/pathogenicity , Animals , Anti-Bacterial Agents/pharmacology , Coinfection/genetics , Coinfection/immunology , Coinfection/veterinary , Cytokines/genetics , Drug Resistance, Bacterial , Fish Diseases/genetics , Fish Diseases/immunology , Fish Diseases/pathology , Fish Proteins/genetics , Gram-Negative Bacterial Infections/genetics , Gram-Negative Bacterial Infections/immunology , Gram-Negative Bacterial Infections/pathology , Gram-Negative Bacterial Infections/veterinary , Kidney/pathology , Liver/pathology , Muscles/pathology , RNA, Messenger/metabolism , RNA, Ribosomal, 16S/genetics , Virulence/genetics , Zebrafish/genetics , Zebrafish/immunology , Zebrafish/microbiology
18.
Fish Shellfish Immunol ; 76: 240-246, 2018 May.
Article in English | MEDLINE | ID: mdl-29510255

ABSTRACT

Chitosan nanoparticles (CNPs) were synthesized by ionic gelation method and its immunomodulatory properties were investigated in zebrafish larvae. Average particle size and zeta potential were 181.2 nm and +37.2 mv, respectively. Initially, toxicity profile was tested in zebrafish embryo at 96 h post fertilization (hpf) stage using medium molecular weight chitosan (MMW-C) and CNPs. At 5 µg/mL, the hatching rate was almost similar in both treatments, however, the survival rate was lower in MMW-C compared to CNPs exposure, suggesting that toxicity effect of CNPs in hatched larvae was minimal at 5 µg/mL compared to MMW-C. Quantitative real time PCR results showed that in CNPs exposed larvae at 5 days post fertilization (5 dpf) stage, immune related (il-1ß, tnf-α, il-6, il-10, cxcl-18b, ccl34a.4, cxcl-8a, lyz-c, defßl-1, irf-1a, irf-3, MxA) and stress response (hsp-70) genes were induced. In contrast, basal or down regulated expression of antioxidant genes (gstp-1, cat, sod-1, prdx-4, txndr-1) were observed. Moreover, zebrafish larvae (at 5 dpf stage) exposed to CNPs (5 µg/mL) showed higher survival rate at 72 h post infection stage against pathogenic Aeromonas hydrophila challenge compared to controls. These results suggest that although CNPs can have toxic effects to the larvae at higher doses, CNPs exposure at 5 µg/mL could enhance the immune responses and develop the disease resistance against A. hydrophila, which could be attributed to its strong immune modulatory properties.


Subject(s)
Chitosan/pharmacology , Fish Diseases/immunology , Gram-Negative Bacterial Infections/immunology , Immunity, Innate , Immunologic Factors/pharmacology , Nanoparticles , Zebrafish/immunology , Aeromonas hydrophila/physiology , Animals , Chitosan/administration & dosage , Immunologic Factors/administration & dosage , Longevity , Nanoparticles/administration & dosage , Particle Size
19.
Int J Biol Macromol ; 108: 1281-1288, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29129632

ABSTRACT

In this study, we investigated the antifungal activity and cytotoxicity of ZnO-chitosan nanocomposites (ZnO-C NCs) against Candida albicans and human epithelial type 2 (HEp2) cells, respectively. The crystalline phase, morphology, composition, particle size and optical absorption properties of the synthesized ZnO-C NCs were systematically investigated by various contemporary methods. The X-ray diffraction analysis results showed characteristic diffraction peaks corresponding to both ZnO and chitosan, while field-emission scanning electron microscopy (FESEM) displayed clusters of spherical shaped particulate morphology. UV-vis absorption spectra showed a shift in the optical absorption towards lower wavelength for ZnO-C NCs when compared to ZnO nanoparticles (NPs). The antifungal activity results (against C. albicans) showed that the minimum inhibitory concentration of ZnO NPs and ZnO-C NCs were 200µg/mL and 75µg/mL, respectively, suggesting the greater therapeutic potential of ZnO-C NCs. FESEM analysis results showed the substantial change in the external morphology of C. albicans after treatment with both ZnO NPs and ZnO-C NCs due to the fungal cell membrane damage. ZnO-C NCs displayed lower cytotoxicity with HEp2 cells indicating the good cytocompatibility of the synthesized ZnO-C NCs. It is expected that ZnO and chitosan complement each other and exhibit synergistic effects potential for antimicrobial and biomedical applications.


Subject(s)
Antifungal Agents/chemical synthesis , Antifungal Agents/pharmacology , Candida albicans/drug effects , Chitosan/chemistry , Nanocomposites/chemistry , Zinc Oxide/chemistry , Antifungal Agents/chemistry , Antifungal Agents/toxicity , Chemistry Techniques, Synthetic , Hep G2 Cells , Humans , Microbial Sensitivity Tests
20.
Indian J Microbiol ; 57(4): 427-437, 2017 Dec.
Article in English | MEDLINE | ID: mdl-29151644

ABSTRACT

ABSTRACT: Development of nanostructured films using natural polymers and metals has become a considerable interest in various biomedical applications. Objective of the present study was to develop silver nano particles (AgNPs) embedded chitosan films with antimicrobial properties. Based on the Ag content, two types of chitosan silver nano films, named as CAgNfs-12 (12 mM) and CAgNfs-52 (52 mM) were prepared and characterized. Field emission scanning electron microscope (FE-SEM) images of two CAgNfs showed the circular AgNPs, which were uniformly embedded and distributed in the matrix of chitosan films. Antimicrobial experiment results clearly indicated that CAgNfs can inhibit the growth of fish pathogenic bacteria Vibrio (Allivibrio) salmonicida, V. tapetis, Edwardsiella tarda and fungi Fusarium oxysporum. Moreover, CAgNfs significantly reduced the experimentally exposed V. salmonicida levels in artificial seawater, suggesting that these CAgNfs could be used to develop antimicrobial filters/membranes for water purifying units to eliminate the pathogenic microbes.

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