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Immunostimulatory effect of Recombinant Antimicrobial Peptides

supplemented feed on Salmonid fish against bacterial pathogens


Objectives:
The main aim of this study is to evaluate the immunostimulatory response of pathogenic
bacteria challenged Salmonid fish supplemented with recombinant antimicrobial peptides.
Materials and Methods:
1.

Identification of Antimicrobial Peptides gene in cDNA library:


The antimicrobial peptides which are earlier reported in other fishes, crustaceans and
mammals will be identified from the cDNA library. The identified proteins will be
characterized bioinformatically.

2.

Molecular Cloning:
The identified antimicrobial peptide gene sequence will be cloned in bacterial host and
the cloned antimicrobial peptides will be expressed in expression vectors and purified.
The purified recombinant peptide will be used for further in vitro and in vivo studies

3.

Antimicrobial activity assay of recombinant antimicrobial peptide (in vitro):


The antimicrobial activity of recombinant antimicrobial peptides will be studied against
bacterial pathogens using minimum inhibitory concentration method.

4.

Preparation of AMP supplemented diet:


Four diets containing different dosage of recombinant antimicrobial peptides will be
prepared. One control diet will be maintained without antimicrobial peptide.

5.

Experimental Setup and Challenge:


The fishes will be divided into four groups of 10 each in triplicate and fed with different
dosage of AMPs enriched diet at the rate of 5% of their body weight twice a day.

All

groups will be injected intraperitoneally (i.p.) with 100 l PBS containing bacterial
pathogens at 3.6105 cfu ml1. A group of 25 fishes will be maintained separately for
uninfected control (C) fed with basal diet. At weeks 1, 2, and 4 post-infection, six fishes
will be randomly collected from each tank and anesthetize with MS-222 in de-chlorinated
water for 2 min to collect blood samples for hematological and immunological assays.
6.

Hematological Parameters:
i. Haematocrit
ii. Haemoglobin concentration

iii. Total red blood cell count


iv. Total white blood cells
v. Serum mineral content
vi. Glucose concentration
vii. Total protein content
7.

Histological analysis of gill, liver and spleen

8.

Immunological Assays:
a. Lysozyme activity Assay
b. Super Oxide Dimutase (SOD) Assay
c. Lipid Peroxidation Assay
d. Measurement of fish Immunoglobulin by ELISA

9.

Expression of Antimicrobial peptide gene in various tissues in dietary treatment


with and without infection.

Identification of antimicrobial peptides in Salmonid fish cDNA Library

Cloning of identified antimicrobial peptide

Expression and Purification of recombinant antimicrobial peptide

Invitro antimicrobial activity of AMPs against various bacterial pathogens using minimum
inhibitory concentration technique

The fishes will be fed on four different diets (four different concentrations of recombinant AMPs)
and a control diet for the growth and immunological studies

Each replicate consist of five fishes randomly sampled from a tank at the
beginning, and after 1, 7, 14, and 28 days of AMP feeding

Blood will be collected


from each fish for
hematological studies

Histological analysis of
Liver, gill and spleen

Gene expression activity of


AMP gene in various
tissues

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