Theses and Dissertations
Issuing Body
Mississippi State University
Advisor
Petrie-Hanson, Lora
Committee Member
Pruett, Stephen B.
Committee Member
Jenny, Matthew J.
Committee Member
Pharr, G. Todd
Committee Member
Hanson, Larry
Date of Degree
12-8-2017
Document Type
Dissertation - Open Access
Major
Veterinary Medical Science
Degree Name
Doctor of Philosophy
College
College of Veterinary Medicine
Department
Department of Basic Sciences
Abstract
Rag1-/- mutant zebrafish lack lymphocytes and were used to study the basis of acquired protective immunity in the absence of lymphocytes to the intracellular bacterium Edwardsiella ictaluri. This study morphologically identified and quantified lymphocyte like cells (LLCs) present in the liver, kidney and spleen of these fish. LLCs included Natural Killer (NK) cells and non-specific cytotoxic cells (NCCs) and were discriminated by size, and by the presence of cytoplasmic granules. The antibodies anti-NITR9, anti-NCCRP-1 (5C6) and anti-MPEG-1 were used to evaluate these cell populations by flow cytometry. Gene expression profiles in these tissues were evaluated after the Rag1-/- mutants were intra coelomically injected with the toll like receptor (TLR)-2 ligand, β glucan, TLR3 ligand, Poly I:C, or TLR 7/8 ligand, R848. The genes interferon y (infγ), expressed by activated NK cells and macrophages, tumor necrosis factor α (tnfα), expressed by activated macrophages, myxovirus resistance (mx) expressed by cells induced by IFNα, T-cell transcription factor (t-bet) expressed by NK cells and novel immune type-receptor 9 (nitr-9) expressed by NK cells were evaluated. The TLR ligands induced different patterns of expression and stimulated both macrophages and NK cells. Then fish were vaccinated with an attenuated mutant of E. ictaluri (RE33®) with or without the TLR ligands then challenged with WT E. ictaluri to evaluate protection. RE33® alone and each TLR ligand alone provided protection. Coministration of β glucan and RE33® or R848 and RE33® resulted in survival higher than RE33® alone showing an adjuvant effect. Tissue specific gene expression of ifnγ, t-bet, nitr9, NK cell lysin a (nkla), nklb, nklc and nkld were correlated to protection. The final component of this study was the development of tools to discriminate NK cell populations and evaluate the contribution of macrophages. Rag1-/- zebrafish were modified to express cherry red in lymphocyte like cells using the Lymphocyte specific tyrosine kinase (lck) promotor. Also, rag1-/- zebrafish were modified so that the gene encoding the proto-oncogene serine/threonine-protein kinase that is involved in macrophage training (raf1) is disrupted. This study indicated that the acquired protection in the absence of lymphocytes likely involves NK cells with possible contribution by macrophages.
URI
https://hdl.handle.net/11668/18905
Recommended Citation
Muire, Preeti Judith, "The Characterization of Zebrafish Natural Killer Cells and Their Role in Immunological Memory" (2017). Theses and Dissertations. 4285.
https://scholarsjunction.msstate.edu/td/4285
Comments
rag1-/- mutant zebrafish||immunological memory||Natural killer cells