Bioactiviteit en veiligheid van medicinale planten uit Bangladesh tegen vispathogenen: Een natuurlijke benadering van aquacultuurgezondheid

The surging global population raises critical concerns regarding food security and nutrition, as addressed in the United Nations Sustainable Development Goals (SDGs). Among the food-producing sectors, aquaculture plays a pivotal role in alleviating these challenges by supplying a significant share of animal protein through fish production. As the sector expands rapidly, driven by technological advancements, intensification, and high stocking densities, it is often compromised by farm water quality, making it vulnerable to disease outbreaks. A substantial economic and production loss follows disease outbreaks, particularly in developing countries, like Bangladesh, one of the leading global aquaculture producers of the world. Bacterial pathogens are key contributors to these outbreaks in aquaculture. In response, farmers often rely predominantly on chemotherapeutics and antibiotics due to their affordability and availability in developing countries. Such practices exacerbate the development of antimicrobial resistance (AMR) in aquatic and environmental pathogens and pose food safety concerns. The practice of bulk medication in culture water bodies, combined with a general lack of knowledge regarding antibiotic application among farmers, further increases the risk of residual antibiotic contamination in the human food chain. In this context, sustainable and eco-friendly alternatives are critically needed. Plant derivatives can be a promising alternative due to their relative cost-effectiveness, biodegradability, and broad therapeutic potential. Given Bangladesh's rich plant biodiversity and expanding aquaculture sector, investigating indigenous plants for antibacterial properties presents a viable and eco-friendly strategy for sustainable disease management in aquaculture. This PhD thesis aimed to: (i) assess the current status of finfish aquaculture practices, disease management strategies, and biosecurity measures adoption among fish farmers in Bangladesh; (ii) explore the antibacterial potency of ethnobotanically significant Bangladeshi plants against key fish pathogens; (iii) isolate and characterize bioactive compounds from most promising plant extracts using bioassay guided fractionation approach; and (iv) evaluate the toxicity profile of the selected active extracts in vivo through zebrafish models to support their potential utilization in sustainable aquaculture disease management (Chapter 2). In Chapter 3, a comprehensive survey was conducted to assess the current status of finfish aquaculture practices, disease management strategies, and biosecurity measures adoption among farmers in Bangladesh. The survey involved 400 farmers from six major finfish producing regions of Bangladesh. The findings revealed that the majority were small-scale polyculture (cultures of more than one fish species) operators. While most fish farmers were literate and experienced, only a small portion of the farmers had received formal training or institutional knowledge about farming. Approximately 70% of the farmers reported disease outbreaks on their farms, and the common disease management measures implemented were the use of chemotherapeutics (91%) and antibiotics (53%). Although the overall biosecurity adoption rate was 68%, gaps persisted in the adoption of some critical measures, particularly practices such as access restriction in the farm, avoiding restocking (i.e., not adding new fish during the same production cycle), sharing farm equipment, and following quarantine before stocking, highlighting the need for tailored improvement. Considering the region-based variations in disease management practices and the adoption status of biosecurity measures, this study suggests the development of tailored, need-based biosecurity implementation guidelines, training, and other extension services. Chapter 4 included the preliminary screening of Bangladeshi medicinal plants based on antibacterial potency against key fish bacteria responsible for causing diseases in aquaculture. For this purpose, 55 selected medicinal plants from Bangladesh were collected, and extracts were prepared using four different solvents, including hexane, ethanol, acetone, and water. These extracts were then tested against Aeromonas hydrophila, Pseudomonas fluorescens, Pseudomonas aeruginosa, Streptococcus iniae, and Flavobacterium columnare using the broth microdilution method. The finding revealed that all plants are active against F. columnare and S. iniae with at least one solvent type extract. The antibacterial potency of plant extracts varied with the solvent used for extraction and the tested bacterial strains. The 50% inhibition concentrations (IC50) of the active extracts ranged from 1 to 614 µg/mL, and 35% of the active extracts had IC50 values less than 25 µg/mL. A few plants exhibited a broad spectrum of antibacterial activities against tested pathogens, including Terminalia arjuna, Terminalia bellirica, M. micrantha, Peperomia pellucida, Lawsonia inermis, Phyllanthus emblica, Eryngium foetidum, Amaranthus spinosus, Barringtonia acutangula, Clitoria ternatea, Alstonia scholaris, and Sphagneticola trilobata. Preliminary phytochemical screening revealed the presence of flavonoid, terpenoid, phenolic, glycoside, alkaloid, and steroid phytocompound groups in active plants. In Chapters 5 and 6, the bioassay-guided isolation of active compounds from ethanolic extracts of A. scholaris leaves (AsLEE), T. bellirica fruits (TBfEE), and M. micrantha leaves (MmEE) was conducted using silica-gel column chromatography, high-performance liquid chromatography (HPLC), and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS). These studies resulted in the identification of 4 bioactive compounds. Oleanolic acid was identified in A. scholaris; oleic acid and 9,10,13-trihydroxy-11-octadecenoic acid were identified from T. bellirica; palmitic acid was identified in M. micrantha as a bioactive compound. All identified compounds exhibited a varied range of broad-spectrum antibacterial activities with IC50 values ranging from less than 1 to 191 µg/mL, with oleic acid being the most potent one (< 1 and 2 µg/mL against S. iniae and P. fluorescens, respectively). Zebrafish embryo lethality assays revealed no toxicity effect at concentrations up to 200 µg/mL for palmitic acid, 300 µg/mL for oleanolic acid, and 200 µg/mL for both oleic acid and 9,10,13-trihydroxy-11-octadecenoic acid. These findings suggest that up to the mentioned doses, these compounds are safe for application in zebrafish embryos. However, in chronic exposure and in adults, the dose may have varied effects that need to be investigated. Chapters 5 and 7 evaluated the in vivo developmental and teratogenic toxicity of three promising plant extracts (AsLEE, TBfEE, and MmEE) using zebrafish embryo acute toxicity (FET) assays, in accordance with Organization for Economic Cooperation and Development (OECD) guidelines. Five different concentrations, including 500, 250, 125, 62.5, and 31.25 µg/mL of each plant extract, were used in the FET assays. The half-maximal effective concentrations (EC50) and no-observed-adverse-effect levels (NOAEL) were determined as 254 µg/mL and 62.5 µg/mL for AsLEE; >500 µg/mL and 500 µg/mL for TBfEE; and 142 µg/mL and 31.25 µg/mL for MmEE, respectively. These findings suggest potential development toxicity at higher concentrations, particularly for AsLEE and MmEE. Furthermore, the acute toxicity and sub-acute toxicity of TBfEE and MmEE were evaluated following dietary administration in adult zebrafish (Chapter 7). Acute toxicity was performed following the acute oral toxicity evaluation with the fixed dose method as described in European Union Directive 2004/73/EC, Annex V (Annex 2B), aligning with the OECD Test Guideline (TG) 420. A fixed oral dose of 2000 mg/kg of fish body weight revealed no evident toxicity signs or mortality in both the acute study (once administered) and sub-acute study (exposed for 14 consecutive days) in adult zebrafish. However, the histological evaluation revealed mild alterations in gill, intestine, liver, and kidney tissues in fish of both treatment groups, suggesting that the diet supplementation dose of 2000 mg/kg body weight of both plant extracts has mild inflammatory effects on fish health without having life-threatening consequences. Chapter 8 describes the in vivo evaluation of the antibacterial potency of AsLEE, MmEE, TBfEE, and oleanolic acid (OA, a commercial standard) against A. hydrophila using a zebrafish embryo-larval anti-infection model. Zebrafish embryos were immunosuppressed with 25 µM dexamethasone (2-120 h post fertilization (hpf), except 72-81 hpf), and infected with A. hydrophila at 72 hpf for 9 hours, followed by immersion-based treatment administration (81-120 hpf). Survival was monitored at several time points, and bacterial load clearance was quantified by quantitative real-time polymerase chain reaction (qPCR) analysis. AsLEE demonstrated a non-significant protective effect by improving survival and reducing bacterial load, OA provided transient protection, while TBfEE and MmEE were inactive. The findings underscore the influence of the plant extract's composition and complex mode of action, as well as its solubility, bioavailability, and the model's treatment delivery limitations, highlighting the need for adult fish trials with optimized administration strategies to confirm its therapeutic potential. Overall, this thesis offers insights into current practices and identifies critical gaps in disease management strategies and the adoption of biosecurity measures in Bangladesh's finfish aquaculture sector, while also suggesting the need for tailored and need-based development of biosecurity guidelines and implementation strategies. This study also lays the scientific foundation for the exploration and development of plant-based antibacterial agents as a sustainable alternative strategy in managing fish d