Therapeutic potential of bioactive compounds
for treating fish disease
Nitin Challa
Mentor: Dr. Grace Ramena
Aquaculture
Major protein source
Food and Agriculture Organization of United Nations 2016
Fish Disease Problems Viral Parasitic
Bacterial Fungal
http://www.churchfreedom.org/a-prophecy-regarding-bacteria-important/bacteria/
Columnaris – Flavobacterium columnare
Annelies Maria et al. 2013
https://nextdaykoi.com/koi-fish-facts/your-koi-and-columnaris/
Gram negative
Bacillus
yellow pigmentation
Rhizoid-forming colonies
Columnaris – Symptoms
https://huntfish.mdc.mo.gov/fishing/fish-diseases/columnaris-disease
FDA Approved Treatments
Antibiotics: Oxytetracycline(Terramycin® 200)
Florfenicol (AQUAFLOR)
Chemicals: External Disinfectants Chloramine – T (columnaris)
Hydrogen Peroxide
https://www.fda.gov/AnimalVeterinary/DevelopmentApprovalProcess/Aquaculture/ucm132954.htm
Antibiotic Mode of Action
Target and bind to 30S ribosomal subunit to prevent
aminocyl - tRNA to attach to RNA- ribosome complex, inhibiting protein synthesis
https://amrls.cvm.msu.edu/microbiology/microbiology-module
Bacterial Mechanisms of Resistance
Intrinsic Resistance
Acquired Resistance
https://amrls.cvm.msu.edu/microbiology/microbiology-module
Intrinsic Resistance – Protein Modification
https://amrls.cvm.msu.edu/microbiology/microbiology-module
Intrinsic Resistance – Efflux Pumps
https://amrls.cvm.msu.edu/microbiology/microbiology-module
Intrinsic Resistance – Ribosomal Modification
https://amrls.cvm.msu.edu/microbiology/microbiology-module
Acquired Resistance - Selectivity
https://amrls.cvm.msu.edu/microbiology/microbiology-module
Acquired Resistance - Mutations
https://amrls.cvm.msu.edu/microbiology/microbiology-module
Acquired Resistance – Horizontal Gene Transfer
https://amrls.cvm.msu.edu/microbiology/microbiology-module
Other Alternatives to Drugs
Shoemaker et al., 2011; Olivares-Fuster et al., 2011; Hernanz et al., 1998; Liu et al., 2007; Abdelhamed et al., 2017
Low Efficacy; Time consuming;
Expensive
Natural Bioactive Compounds
https://www.researchgate.net/figure/283338191_fig2_Structural-features-of-plants-and-bioactive-compounds-that-affect-pancreatic-beta-cell
Aromatic plants, Herbs, Spices- Extracts, Essential oils
Antioxidant Activity
Antimicrobial Activity
Bactericidal Activity
Feed Additives Eg. Antioxidants,
Growth Promoters
Food Industry Eg. Flavors, Fragrances,
Preservatives
Cosmetic Industry Eg. Perfumes, Skin Products
Pharmaceutical Industry
Eg. Medicines
Main Activities
Uses
Bacteriostatic Activity
Identification of spices and herbs that have antimicrobial activity against F.Columnare
Bactericidal Effect on F columnare
Bactericidal Effect of Cuminum cyminum
Cuminum cyminum N/A 21.4 N/A N/A
Cuminum cyminum Syzygium aromaticum 20.1 0.1556 <.0001
Cuminum cyminum Thymus vulgaris 19.6 0.1556 <.0001
Cuminum cyminum Cinnamomum verum 18.3 0.1556 <.0001
Cuminum cyminum Allium sativum 16.7 0.1556 <.0001
Cuminum cyminum Coriandrum sativum 16.4 0.1556 <.0001
Cuminum cyminum Salvia officinalis 16.2 0.1556 <.0001
Cuminum cyminum Allium cepa 13.3 0.1556 <.0001
Cuminum cyminum Zingiber officinale 13.2 0.1556 <.0001
Cuminum cyminum Anethum graveolens 11.3 0.1556 <.0001
Cuminum cyminum Curcuma longa 10.2 0.1556 <.0001
Cuminum cyminum Terramycin® 200 19.7 0.1556 <.0001
Cuminum cyminum Methanol 0 0.1556 <.0001
Dunnet's Test
Extract compared Extract/AntibioticMean Inhibition
(mm)
Pooled Standard
ErrorP- Value
Degrees of Freedom = 51
Terramycin®
200 Cuminum cyminum 21.4 0.1651 <.0001
Terramycin®
200 Syzygium aromaticum 20.1 0.1651 0.0503
Terramycin®
200 Thymus vulgaris 19.6 0.1651 1.0
Terramycin®
200 Cinnamomum verum 18.3 0.1651 <.0001
Terramycin®
200 Allium sativum 16.7 0.1651 <.0001
Terramycin®
200 Coriandrum sativum 16.4 0.1651 <.0001
Terramycin®
200 Salvia officinalis 16.2 0.1651 <.0001
Terramycin®
200 Allium cepa 13.3 0.1651 <.0001
Terramycin®
200 Zingiber officinale 13.2 0.1651 <.0001
Terramycin®
200 Anethum graveolens 11.3 0.1651 <.0001
Terramycin®
200 Curcuma longa 10.2 0.1651 <.0001
Terramycin®
200 Terramycin® 200 19.7 N/A N/A
Terramycin®
200 Methanol 0 0.1651 <.0001
Dunnet's Test
Degrees of Freedom = 51
Antibiotic compared ExtractMean
Inhibition (mm)
Pooled
Standard ErrorP- Value
Bactericidal Effect of OTC Compared to Extracts
Determine the Minimum Inhibitory and compare the activity to Oxytetracycline
Bactericidal Effect of Extracts at increasing Concentrations
Minimum Inhibitory Concentration of OTC
Syzygium aromaticum Syzygium aromaticum 15.08 N/A N/A
Syzygium aromaticum Cuminum cyminum 9.1 0.1581 <.0001
Syzygium aromaticum Thymus vulgaris 9.7 0.1581 <.0001
Syzygium aromaticum Cinnamomum verum 9.3 0.1581 <.0001
Syzygium aromaticum Allium sativum 4.6 0.1581 <.0001
Syzygium aromaticum Coriandrum sativum 6.2 0.1581 <.0001
Syzygium aromaticum Salvia officinalis 8.9 0.1581 <.0001
Syzygium aromaticum Allium cepa 7.54 0.1581 <.0001
Syzygium aromaticum Zingiber officinale 8.8 0.1581 <.0001
Syzygium aromaticum Anethum graveolens 2.7 0.1581 <.0001
Syzygium aromaticum Curcuma longa 4.9 0.1581 <.0001
Syzygium aromaticum Terramycin®
200 9.8 0.1581 <.0001
Syzygium aromaticum Methanol 0 0.1581 <.0001
Mean Inhibition
(mm)
Dunnet's Test - Comparison of Syzygium aromaticum with other extracts (5mg) and
Terramycin® 200 (5 µg)
Degrees of Freedom = 47.1
Extract compared Extract/AntibioticPooled
Standard ErrorP- Value
Bactericidal Effect of Syzygium aromaticum
Summary
1. All the Eleven extracts tested had Bactericidal effect against F.columnare.
2. Cumin had highest bactericidal effect when compared to other extracts and the antibiotic.
3. Clove and Thyme had similar antimicrobial effect like the antibiotic.
4. Although Cumin showed higher antimicrobial activity at the highest concentration, Clove had the best effect at MIC tested.
Current Status of The Project
Dr. Grace Ramena
Mentor Dr. Muthusamy Manoharan Dr. Lin Xie
Advisory Committee Members