The four cattle were housed together at approved WSU and USDA-ARS research barns in Moscow, ID and Pullman, WA for the duration of this study. dexamethasone were administered intravenously to all four cattle. Within two weeks of immunosuppression, all animals developed viral neutralizing antibody responses to BoHV-1, and all animals maintained BVDV viral neutralizing CC-671 capacity. Overall, nebulization of BoHV-4-A-CMV-IgK-gE2gD-TM persistently infects cattle, is capable of eliciting antigen-specific immunity following immunization, including in the presence of pre-existing BoHV-4 immunity, and recrudescence of the virus boosts the immune response CC-671 to BoHV-4-vectored antigens. These results indicate that BoHV-4 is a viable and attractive vaccine delivery platform for use in cattle. Introduction Bovine herpesvirus-4 (BoHV-4) is within the rhadinovirus genus in the gammaherpesvirus subfamily of the Herpesviridae family . As is usually typical of other gammaherpesviruses, BoHV-4 initially infects mucosal epithelial cells, and replicates and undergoes latency in monocytes and macrophages . BoHV-4 has been isolated from a variety of tissues from healthy cattle [3, 4]. Although BoHV-4 has also been identified in diseased respiratory, enteric, and most often, reproductive tissues, causal associations with disease have not been definitively established, and experimental contamination of cattle does not reliably result in overt clinical disease [5, 6]. Herpesviruses have intrinsic properties that make them attractive for use as viral vaccine vector candidates. Herpesviruses are capable of incorporating large amounts of foreign genetic material and can undergo replication-competent persistent infections in the host. For CC-671 example, herpesvirus of turkeys (HVT) has been developed as successful, single dose veterinary vaccine capable of producing long term protective responses against avian influenza in chickens based in part on the ability to undergo persistent infection, even in the presence of neutralizing maternal antibodies [7C10]. Thus herpesvirus vectors are thought to be capable of priming and maintaining lifelong antigen-specific protective immunity. In addition to these intrinsic biological properties of herpesviruses, BoHV-4 is usually non-oncogenic and avirulent under experimental challenge conditions . In light of these properties, BoHV-4 has been successfully developed as a veterinary vaccine vector in model animal species against a variety of infectious diseases. BoHV-4-vectored vaccines have successfully induced neutralizing antibody responses to bovine herpesvirus-1 (BoHV-1) and Bovine Viral Diarrhea Virus (BVDV) type 1 in rabbits [11, 12], neutralizing antibody responses to BVDV type 1 in sheep , neutralizing antibody responses to Bluetongue virus and to Peste de Petits Ruminants (PPR) virus in mice, respectively [14, 15], and protective viral neutralizing antibody responses against caprine herpesvirus-1 in goats . Despite the success of the BoHV-4 platform in these non-BoHV-4 host species, and the intrinsic benefits of the BoHV-4 platform, immunization using BoHV-4 has never been attempted in cattle. The seroprevalence of BoHV-4 in cattle populations varies widely in different geographical regions; recently, common global seroprevalence estimates vary between 4C30% . Some dairy herds experience up to 88% seroprevalence  and wild African buffalo populations reportedly have up to 94% seroprevalence . Given the seroprevalence of BoHV-4 in most bovid populations, it is critical to determine whether pre-existing immunity directed at the vector could preclude the development of antibody responses HAS2 to the vaccine antigen after BoHV-4-vectored immunization. This is a concern with other viral vaccine delivery systems, particularly with the use of adenoviral vectors . Specifically, reduction in recombinant adenoviral vaccine vector efficiency attributed to decreased transgene transduction has been described following vaccination of humans that had pre-existing neutralizing antibodies developed to circulating strains of adenovirus . The immunodominant BVDV type 1-derived antigen E2 and BoHV-1-derived glycoprotein D were used as the model immunogens, and were expressed as a chimeric protein by recombinant BoHV-4 (BoHV-4-A-CMV-IgK-gE2gD-TM). This recombinant BoHV-4 construct expressing E2 and gD has been.