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