Search results for: lysozyme

Authors: S. F. Afzali, W. L. Wong

Abstract:

The epizootic ulcerative syndrome (EUS) causes by the oomycete fungus, Aphanomyces invadans; known to be one of the infectious fish diseases for farmed and wild fishes in fresh and brackish-water from the Asia-pacific region, America and Africa. Although, EUS had been documented by the Office International des Epizooties (OIE) since 1995, hitherto, there is neither standard chemical agents that can be used for successful treatment of this destructive infection in the time of outbreak; nor available vaccine for prevention. Plant-based remedies in controlling fish diseases are gaining much attention recently as an alternative to chemical treatments, which possess negative effects to the environment and human. In present study, Sonneratia alba, a mangrove plant belongs to the Sonneratiaceae family, was screened in vitro and in vivo for its antifungal activity against A. invadans mycelium growth and its effects on fish innate immune system and disease resistant. The in vitro tests was performed using the disc diffusion methods with measurements of minimum inhibitory concentration (MIC) and inhibition zone. For in vivo study, the S. alba extract supplemented diets were administrated at 0.0, 1.0%, 3.0%, and 5.0% on healthy goldfish, Carassius auratus, which challenged with A. invadans zoospores (100 spores/ml). To compare the significant differences in the hematological and immunological parameters obtained from the experiments, the data were analysed using the SPSS. The methanol extract of S. alba effectively inhibited the mycelial growth of A. invadans at a minimum concentration of 1000 ppm for agar and filter paper diffusion experiments. In the agar diffusion test, 500 ppm of the extract inhibited the fungus mycelial growth up to 96 hours after exposure. The mycelial growth from the edge of the pre-inoculated A. invadans agar discs treated with S. alba extracts at concentrations of 100, 500 and 1000 ppm were 15, 8 and 0 mm respectively. The results of the filter paper disc test showed that the S. alba extract at its minimal inhibitory concentration (1000 ppm) has similar qualitative inhibitory effect as malachite green at 1 ppm and formalin at 250 ppm. According to the in vivo tests findings, in the infected fish fed with 3.0% and 5.0% supplementation diet, the numbers of white blood cell and myeloperoxidase activity significantly increased after the second week of treatment. Whilst the numbers of red blood cell significantly decreased in the infected fish fed with 0.0 and 1.0% supplementation diet. After the third week of feeding, significant increases in the total protein, albumin level, lysozyme activity were recorded in the infected fish fed with 3.0% and 5.0% supplementation diet. Also, the enriched diets increased the survival rate as compared to the untreated group that suffered from 90% mortality. The present study indicated that S. alba extract may inhibit the mycelial growth of A. invadans effectively, suggesting an alternative to other chemotherapeutic agents, which brought much environmental and health concerns to the public, for EUS treatment.

Keywords: fungal pathogen, goldfish, organic extract, treatment

Procedia PDF Downloads 256

Authors: Shikha Rani, Neeta Sehgal, Vipin Kumar Verma, Om Prakash

Abstract:

Introduction: Fish is an important protein source for humans and has great economic value. Fish cultures are affected due to various anthropogenic activities that lead to bacterial and viral infections. Aeromonas hydrophila is a fish pathogenic bacterium that causes several aquaculture outbreaks throughout the world and leads to huge mortalities. In this study, plants of no commercial value were used to investigate their immunostimulatory, antioxidant, anti-inflammatory, anti-bacterial, and disease resistance potential in fish against Aeromonas hydrophila, through fish feed fortification. Methods: The plant was dried at room temperature in the shade, dissolved in methanol, and analysed for biological compounds through GC-MS/MS. DPPH, FRAP, Phenolic, and flavonoids were estimated following standardized protocols. In silico molecular docking was also performed to validate its broad-spectrum activities based on binding affinity with specific proteins. Fish were divided into four groups (n=6; total 30 in a group): Group 1, non-challenged fish (fed on a non-supplemented diet); Group 2, fish challenged with bacteria (fed on a non-supplemented diet); Group 3 and 4, fish challenged with bacteria (A. hydrophila) and fed on plant supplemented feed at 2.5% and 5%. Blood was collected from the fish on 0, 7th, 14th, 21st, and 28th days. Serum was separated for glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase assay (ALP), lysozyme activity assay, superoxide dismutase assay (SOD), lipid peroxidation assay (LPO) and molecular parameters (including cytokine levels) were estimated through ELISA. The phagocytic activity of macrophages from the spleen and head kidney, along with quantitative analysis of immune-related genes, were analysed in different tissue samples. The digestive enzymes (Pepsin, Trypsin, and Chymotrypsin) were also measured to evaluate the effect of plant-supplemented feed on freshwater fish. Results and Discussion: GC-MS/MS analysis of a methanolic extract of plant validated the presence of key compounds having antioxidant, anti-inflammatory, anti-bacterial, anti-inflammatory, and immunomodulatory activities along with disease resistance properties. From biochemical investigations like ABTS, DPPH, and FRAP, the amount of total flavonoids, phenols, and promising binding affinities towards different proteins in molecular docking analysis helped us to realize the potential of this plant that can be used for investigation in the supplemented feed of fish. Measurement liver function tests, ALPs, oxidation-antioxidant enzyme concentrations, and immunoglobulin concentrations in the experimental groups (3 and 4) showed significant improvement as compared to the positive control group. The histopathological evaluation of the liver, spleen, and head kidney supports the biochemical findings. The isolated macrophages from the group fed on supplemented feed showed a higher percentage of phagocytosis and a phagocytic index, indicating an enhanced cell-mediated immune response. Significant improvements in digestive enzymes were also observed in fish fed on supplemented feed, even after weekly challenges with bacteria. Hence, the plant-fortified feed can be recommended as a regular feed to enhance fish immunity and disease resistance against the Aeromonas hydrophila infection after confirmation from the field trial.

Keywords: immunostimulation, antipathogen, plant fortified feed, macrophages, GC-MS/MS, in silico molecular docking

Procedia PDF Downloads 55