Bovine herpesvirus-1 (BoHV-1) causes recurrent respiratory and genital infections in cattle; and predisposes them to lethal secondary bacterial infections. Vaccination is a primary strategy to prevent and reduce the severity of disease associated with BoHV-1, and to reduce virus transmission. While modified live (MLV) or killed (KV) BoHV-1 vaccines exist, these are expensive to produce, can cause disease (MLV) or may be ineffective (KV). Development of a DNA vaccine for BoHV-1 has the potential to address these shortcomings, but the very small amount of antigen expressed after DNA immunization presents a barrier to successful immunization of large animals. Engineering the vaccine to target this limited quantity of antigen to dendritic cells (DCs), the cells that prime immune responses, by attracting immature DCs (iDCs) to the vaccination site, is one way that DNA vaccine efficacy might be improved. Beta (β)-defensins are chemotactic peptides that, in studies with mice, improve induction of immune responses to DNA vaccines and this is due, at least in part, to their ability to attract iDCs to the site of vaccination. Accordingly, the objective of the studies described in this thesis was to determine whether using a bovine β-defensin in a DNA vaccine would enhance immune responses to the vaccine and subsequently protect cattle upon challenge with BoHV-1. First I characterized the bovine iDC and then used these cells to screen a panel of synthesized bovine β-defensins for chemotactic activity. The results showed that bovine neutrophil β-defensin (BNBD) 3, BNBD9 and enteric β-defensin (EBD) were equally the most chemotactic of the fourteen synthesized peptides for bovine iDCs. Because BNBD3 is the most abundant of the thirteen BNBDs and was able to attract CD1+ DCs when injected into the skin, I chose BNBD3 as the peptide I would use for the rest of the project. Next I constructed plasmids that expressed BNBD3; either alone or as a fusion construct with the BoHV-1 antigen truncated glycoprotein D (tgD), and then tested the effects of the plasmids as vaccines in both mice and cattle. In cattle, the addition of BNBD3 as a fusion strengthened the Th1 bias and increased cell-mediated immune responses to the DNA vaccine but not antibody response or protection from BoHV-1 infection. Given that inefficient humoral immune responses have been implicated in a lack of protection from BoHV-1 challenge, these results suggested that the successful BoHV-1 DNA vaccine would need to induce a much stronger humoral response. Lastly I assessed the ability of BNBD3 to improve humoral responses to pMASIA-tgD when complexed with the DNA vaccine and found that the vaccine complexed at a nanomolar peptide to DNA ratio of 125:1 increased humoral responses of mice. In vitro, treatment of mouse bone-marrow DCs with BNBD3 induced phenotypic and functional maturation/activation. This is an important aspect for vaccination in the skin, since after uptake, the DC must “mature” in order to traffic from the site of vaccination to the draining lymph node where induction of antigen-specific responses, by activated DCs, takes place. The findings in this thesis show that bovine β-defensins are chemotactic for bovine iDCs. I also show that using a bovine β-defensin as a fusion construct in a DNA vaccine enhances cell mediated but not humoral responses of cattle and yet this vaccine is protective against BoHV-1 challenge. I demonstrate that a bovine β-defensin, when used as a peptide to complex an antigen-encoding plasmid, can increase humoral responses. My work shows a multifunctional ability of bovine β-defensins to modulate and increase immune responses and suggests that bovine β-defensins likely have further untapped potential to enhance efficacy of DNA vaccines for large animals.