Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
Infectious Diseases
Non Technical Summary
Vaccines controlling viral, bacterial and protozoal infections in farm-raised fish are an effective way to prevent infectious diseases and reduce economic losses in aquaculture. Vaccination elicits memory T and B cells that protect against infection. The major goal of our work is to determine the stability of memory B and T cell populations in channel catfish after infection with the protozoan parasiteIchthyophthirius multifiliis(Ich) or vaccination with protective Ich antigens, referred to as i-antigens (i-ags).The objectives of the project are to determine: 1) the DNA sequences encoding the CDR3 domains for the B cell receptor IgM heavy chain (IGH) and for the T cell receptor beta chain (TCR beta) from clonally expanded catfish B and T cell populations responding to vaccination or infection; 2) if the percentage of these specific CDR3 sequences in B and T cell populations changes over 3 years; 3) if repeated vaccination with i-antigen or a non-related antigen changes their relative percentage. We will use in-depth illumina MiSeq DNA sequencing to define CDR3 sequences for i-ag correlated TCR beta and IgH transcripts. Each read will cover 250 bases. A single run will generate approximately 1 x 107 reads. This will allow statistical analysis of CDR3 sequences in B and T cell populations.This project will determine how the immune system of fish maintains homeostasis in the face of disease through the analysis of anticipatory and immune repertoires of T and B cells in channel catfish. This work will have impact on disease prevention in farm-raised food fish through vaccine improvement and enhanced adaptive immune responses. This work will contribute to the economic production of healthy and disease-free fish in the United States.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
0%
Goals / Objectives
The major goal of this project is to determine the stability of both memory B and T cell populations in channel catfish following vaccination with i-antigens or infection with the pathogenic ciliate Ichthyophthirius multifiliis (Ich). The hypothesis of the research is that populations of memory B and T cells recognizing Ich antigens can be defined experimentally and tracked by their specific CDR3 sequences.
Project Methods
Our objective is to measure T cell receptor CDR3 diversity of T and B cells in head kidney and spleen (central lymphoid organs); and PBL (circulating lymphocytes), skin and fins (peripheral tissues and sites of Ich infection). We will construct cDNA libraries from total RNA isolated from PBL, fin, skin, head kidney and spleen of channel catfish. Using an Illumina platform, we will sequence in-depth regions of approximately 250 base pairs extending from the V gene, trhough the D and J genes and into the conservied C gene, which encompasse the CDR3 domains. The CDR3 spans approximately 35 to 45 bases and is defined as the region between the second conserved cysteine of the V gene and the conserved phenylalanine in the TCR beta gene, or the conserved tryptophan in the IgH J gene. A distinct TCR beta or IgH sequence is defined as having an in-frame VDJ rearrangement that encodes the CDR3 domain of the receptor, including modifications at the DV and DJ junctions, and flanking V and J genes. The D gene is contained within the CDR3 region.We will use channel catfish infected with Ich or vaccinated with i-antigen. The fish will be separated into the following experimental groups:A) Ich-infected fish (A-1 uninfected control; A-2 infected and immune)B) I-antigen vaccinated fish (B-1 PBS vaccinated control; B-2 i-antigen vaccinated; B-2ch i-antigen vaccinated and challenged with Ich at 2 years; B-3 i-antigen vaccinated and boosted with i-antigen at 6, 12, and 18 months; B-3ch i-antigen vaccinated and boosted with i-antigen at 6, 12, and 18 months and challenged with Ich at 2 years; B-4 i-antigen vaccinated and boosted with BSA at 6, 12, and 18 months.