Consequences of exposure to cattle persistently infected with BVDV on viral replication and surface expression of bovine peripheral blood mononuclear cells infected in vitro with BVDV and BHV-1

Cattle industry is of great importance for the Italian and Spanish economy, being considered one of the most important economic sectors related to livestock production in these countries, and in productive importance within the European Union. Consequently, a wide knowledge of the diseases affecting this industry becomes critical in order to improve the control, and thus, the productivity. The aim of this study was to develop an experimental model for understanding the mechanisms of immunomodulation responsible for the state of immunosuppression induced by bovine viral diarrhea virus (BVDV) in the herds and its role in the establishment of the Bovine Respiratory Disease Complex, assessing the responsiveness of peripheral blood mononuclear cells (PBMCs) from BVDV-free animals in comparison with animals in contact with an immunotolerant calf to BVDV, both subjected to in vitro infectons with BVDV and/or bovine herpesvirus (BHV-1). For this work, sera and PBMCs were collected from eight heifers, four from a seronegative dairy herd and four from other suffering an ongoing infection due to contact with a persistently infected (PI) animal to BVDV in Lecco and Como provinces. PBMCs were separated into 4 groups of infection: Uninfected control, infected with noncytopathogenic BVDV-1a, infected with BHV-1 subtype 1 and infected with both viruses, being incubated for 18, 24, 48, and 72 hours. To quantify intracellular and extracellular BVDV and BHV-1 in PBMC cultures, a sensitive quantitative PCR method was devised. Replicative capacity of viruses and their elimination to the extracellular environment were evaluated by measuring extracellular viral titres. To determine the effect of these infections on the expression of different leukocyte differentiation antigens, flow cytometry analyses of PBMCs were carried out. At the moment of study, animals from the free-herd were confirmed to be BVDV and BHV-1 antigen and antibody free, while animals exposed to the PI calf were confirmed to be BVDV and BHV-1 antigen free and with BVDV neutralization antibody titres >128. Results showed that PBMCs of all animals were efficiently infected with BVDV and/or BHV1 in the in vitro conditions tested, being observed that animals in contact with the PI calf in the herd were more susceptible to the infections, especially due to BHV-1 elimination to the extracellular environment, thus favouring the infection of other cell populations. This study revealed an absence of changes in the percentage of T lymphocytes despite the viral infections in both groups of animals, being observed only a CD25 up-regulation that affects animals in contact with the PI calf, which possibly was due to the process of immunization itself. The main findings in the PBMCs subpopulations included the reduced percentage of monocytes in the animals in contact with the PI animal after in vitro infection with BHV-1, which also produces a detrimental effect on CD11b expression in these cells, reflecting synergic mechanisms that undermine the response of monocytes-macrophages and, in turn, the innate immune response to these viruses. Monocytes also seemed to down-regulate CD80 expression in response to BHV-1 infection, a fact that may be responsible for an impaired process of antigen presentation and activation of lymphocytes.