The objective of this study was to evaluate and characterize the vascular changes observed in the lungs of healthy calves and calves with subclinical BVD both experimentally inoculated with BHV-1.1, and to clarify the role of the pulmonary MΦs in the local response to the secondary pathogen.
The results show that following BHV-1 inoculation, both groups of calves displayed a mononuclear cell infiltrate and prompted major vascular alterations in the lungs, marked by intense intravascular coagulation in small and medium-sized blood vessels from an early stage of the disease. Three mechanisms, jointly known as Virchow’s triad, can induce thrombosis : 1) endothelial wall injury; 2) abnormalities of blood constituents (platelets, coagulation and fibrinolytic pathways); and 3) abnormalities of blood flow.
With regard to endothelial damage, infection by human herpes simplex virus – belonging to the same subfamily that BHV-1  – has been shown to cause endothelial injury, favoring the exposure of subendothelial tissue and the release of procoagulant mediators [45, 46]. Here, however, neither histopathological nor ultrastructural examination disclosed any morphological evidence of endothelial damage in either of the inoculated groups.
Analysis of the cell components involved in coagulation pathways revealed fibrin deposits and intense platelet aggregation in the pulmonary microvasculature of both groups; these findings were particularly marked in the BVDV/BHV-1 group at 4 dpi, coinciding with a significant increase of the rectal temperature and severe clinical respiratory symptoms . According to this, in the course of certain acute viral infections, platelets may be activated in vivo, leading to their degranulation, aggregation and withdrawal from circulation [47–49]. The procoagulant activity of BVDV and BHV-1 has been reported in vitro . In our experimental study, it was increased in the co-infected calves due to the concomitance of both agents in the lungs between 2 and 7 dpi. Despite the absence of BVDV in the blood of these calves at the moment of BHV-1 inoculation, it does not completely disappear, being detected by PCR in the lungs and by IHC in lymphoid tissues throughout the experience . However, there was no evidence in any stage of direct interaction between these viruses and platelets, which would suggest that platelet activation may be enhanced by indirect mechanisms including the expression of inflammatory mediators released by MΦs, which are known to play a major role in the maintenance of tissue homeostasis [11, 52].
The study of pro-inflammatory cytokines revealed alterations in the kinetics and magnitude of TNFα and IL-1 expression; both mediators can prompt changes in coagulation by increasing the number of endothelial adhesion molecules or increasing vascular permeability [11, 53]. Thus, the BVH-1 group calves display an increase in IL-1 synthesis by septal MΦs coinciding with the onset of platelet aggregation in the lungs (2 dpi). This, together with the subsequent action of TNFα, would favor the maintenance of the procoagulant setting. By contrast, BVDV/BHV-1 group calves, whilst exhibiting a higher number of IMΦs – the main producers of these cytokines – displayed inhibited IL-1 expression until 7 dpi, along with a minimal TNFα response. This impaired cytokine production, also observed at the systemic level , indicates that the synergic action of both chemical mediators can be ruled out as a potential mechanism for inducing platelet aggregation in the co-infected group.
Calves inoculated only with NCP BVDV (0 dpi for the BVDV/BHV-1 group) displayed greater expression of iNOS by septal MΦs than healthy calves [34, 35, 54]. However, following BHV-1 inoculation, BVDV/BHV-1 group calves exhibited an early decline in iNOS (1 dpi), an inflammatory mediator that limits the extent and duration of pathogen-induced platelet activation . This finding, together with the moderate response of TNFα, may have favored the appearance of platelet aggregates in the early stages of the disease, and intense aggregation coinciding with the greatest decrease in iNOS levels (4 dpi).
The intense platelet aggregation observed in the lung microvasculature of the BVDV/BHV-1 group at 4 dpi, together with the increase in number and size of PIM as a result of phagocytic and secretory activation would indirectly prompt a slowdown in blood flow and a subsequent response by the COX-2 enzyme aimed at reversing that process . However, in view of the damage observed at later stages, this action was presumably unable to counter the procoagulant events associated with the drop in iNOS expression, these being additionally enhanced by the delayed action of IL-1 in co-infected animals. Slowed blood flow, together with cytokine release, may lead to increased vascular permeability and extravasation of leukocytes into the pulmonary parenchyma [11, 53, 56, 57].
Therefore, the results of this study indicate that the concomitance of BVDV and BHV-1 in the lungs enhances a synergic action of their pathogenic mechanisms, disrupting the maintenance of pulmonary homeostasis by facilitating the establishment of an inflammatory and procoagulant environment, characteristic of the BRDC, which appears to be modulated by inflammatory mediators released by pulmonary MΦs. In this respect, further research is required into the possible involvement of this concomitance, through the use of live BVDV and BHV-1 vaccines, in the triggering of an impaired pulmonary immune response. On the contrary, animals pre-inoculated with BVDV - despite suffering a transient infection - exhibit an alteration in the response of pro-inflammatory cytokines which play a key role in activating the immune response.