Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Animal Science

First Advisor

Jason Botten


The goal of the work presented herein was to further our understanding of Bovine Leukocyte Antigen (BoLA) class I diversity of Holstein cattle and develop tools to measure class I restricted T cell responses to intracellular pathogens such as foot and mouth disease virus (FMDV) following vaccination. BoLA is a highly polymorphic gene region that allows the bovine immune system to differentiate pathogen-infected cells from healthy cells. Immune surveillance by CD8+ T cells plays an important role in clearing viral infections. These CD8+ T cells recognize BoLA class I molecules bearing epitopes (antigenic peptides) of intracellular origin in their peptide binding groove. Polymorphisms in the peptide binding region of class I molecules determine affinity of peptide binding and stability during antigen presentation. Different antigen peptide motifs are associated with specific genetic sequences of class I molecules. In order to better understand the adaptive immune response mediated by BoLA molecules, technologies from human medicine such as high-throughput sequencing, biochemical affinity and stability assays, tetramers and IFN-γ ELIspot assays could be applied. Therefore, it was hypothesized that we can translate these technologies from the study of human T cell responses to the study of cattle immunity.

The first objective was to establish a comprehensive method for genotyping BoLA of Holstein cattle by using Illumina MiSeq, Sanger sequencing and polymerase chain reaction sequence-specific primers (PCR-SSP) (See Chapter 2). This is an important first step in order to study the BoLA restricted immune responses following FMDV vaccination. The second objective was to define the FMDV capsid protein peptide repertoire bound by BoLA class I molecules using bioinformatics and biochemical affinity and stability assays to facilitate the identification of T cell epitopes (See Chapter 3). The third objective was to demonstrate clonal T cell expansion for specific epitope polypeptides using ex-vivo multi-color flow cytometric MHC-epitope complexes (tetramers), followed by IFN-γ production measured by an ELIspot assay to quantify and define the antigen specific response of Holstein cattle to FMDV vaccination (see Chapter 4). In this, my dissertation studies aimed to improve our understanding of the BoLA class I restricted T-cell responses to candidate FMDV vaccines in Holstein cattle. In this manner, my research will improve animal health through the production of assays for characterizing the bovine immune response to intracellular pathogens and enhance vaccine design leading to improved biologicals to protect cattle from devastating infectious diseases.



Number of Pages

186 p.