Pathogenic mechanisms implicated in the intravascular coagulation in the lungs of BVDV-infected calves challenged with BHV-1
© Risalde et al.; licensee BioMed Central Ltd. 2013
Received: 10 July 2012
Accepted: 18 February 2013
Published: 18 March 2013
Resistance to respiratory disease in cattle requires host defense mechanisms that protect against pathogens which have evolved sophisticated strategies to evade them, including an altered function of pulmonary macrophages (MΦs) or the induction of inflammatory responses that cause lung injury and sepsis. The aim of this study was to clarify the mechanisms responsible for vascular changes occurring in the lungs of calves infected with bovine viral diarrhea virus (BVDV) and challenged later with bovine herpesvirus type 1 (BHV-1), evaluating the role of MΦs in the development of pathological lesions in this organ. For this purpose, pulmonary lesions were compared between co-infected calves and healthy animals inoculated only with BHV-1 through immunohistochemical (MAC387, TNFα, IL-1α, iNOS, COX-2 and Factor-VIII) and ultrastructural studies. Both groups of calves presented important vascular alterations produced by fibrin microthrombi and platelet aggregations within the blood vessels. These findings were earlier and more severe in the co-infected group, indicating that the concomitance of BVDV and BHV-1 in the lungs disrupts the pulmonary homeostasis by facilitating the establishment of an inflammatory and procoagulant environment modulated by inflammatory mediators released by pulmonary MΦs. In this regard, the co-infected calves, in spite of presenting a greater number of IMΦs than single-infected group, show a significant decrease in iNOS expression coinciding with the presence of more coagulation lesions. Moreover, animals pre-inoculated with BVDV displayed an alteration in the response of pro-inflammatory cytokines (TNFα and IL-1), which play a key role in activating the immune response, as well as in the local cell-mediated response.
The bovine respiratory disease complex (BRDC) is an important problem for the cattle industry, often resulting in severe economic losses [1, 2]. This fatal bovine respiratory infection is a multi-factorial disease associated with a primary viral infection followed by a secondary bacterial infection. It is frequently characterized by concurrent infections of several pathogens. The etiologic agents related to feedlot pneumonias include bovine viral diarrhea virus 1 and 2 (BVDV-1 and BVDV-2), bovine herpesvirus type 1 (BHV-1), bovine parainfluenza-3 virus, bovine respiratory syncytial virus, bovine adenovirus, bovine coronavirus, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and Mycoplasma spp. [3–6]. Resistance to respiratory disease in cattle requires host defense mechanisms that protect against viral and bacterial pathogens that have also evolved sophisticated strategies to evade the host immune responses, including among others an altered pulmonary macrophages (MΦs) function or the induction of profound inflammatory responses that cause lung injury and sepsis .
The inflammatory process is a protective mechanism that occurs in response to trauma, infection or tissue injury [7, 8]. Increased blood supply, enhanced vascular permeability and migration of immune cells occur at damaged sites. In this process, MΦs play a central role in managing different immunopathological phenomena through the secretion of inflammatory mediators such as nitric oxide (NO), prostaglandins, and the pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin (IL)-1 [9–11].
NO is a potent vasodilator, acting to maintain vascular tone and function within the vessel wall, generated from L-arginine by nitric oxide synthase (NOS) enzymes. There are three main isoforms of NOS with distinct functions and patterns of expression: endothelial NOS, neuronal NOS and inducible NOS (iNOS) highly expressed in MΦs [10, 12].
Prostaglandins are other important inflammatory mediators implicated in the vascular homeostasis since its low levels can prevent or reverse aggregation of platelets and induce vasodilation [9, 13]. These molecules are produced from arachidonic acid metabolites by the catalysis of cyclooxygenase-2 (COX-2) .
Pro-inflammatory cytokines induce integrin expression and redistribution of leukocytes, which contribute to their recruitment and activation at the site of inflammation [15, 16], as well as increasing vascular permeability and tissue injury [17, 18]. There is evidence that inflammatory cytokines are the main orchestrators of the inflammatory cascade in BRDC, detecting high levels of TNFα and IL-1 in the airways of cattle infected with respiratory pathogens [19–22].
BVDV is a pestivirus that although it is not a primary agent in the pathogenesis of BRDC, it can suppress the host immune system and increase the risk of secondary infections, thus enhancing pulmonary colonization by other pathogens such as BHV-1 [23–25]. The mechanisms of the immunosuppressive action of BVDV are object of debate, including changes related to decreased lymphocyte proliferation [26, 27], severe lymphoid depletion in lymphoid tissues [28–30], decreased chemotaxis and phagocytic activity , increased production of prostaglandin E2 [32, 33], increased NO synthesis in response to lipopolysaccharide [34, 35] and impaired production of pro-inflammatory cytokines [30, 36–39].
Therefore, the aim of this study was to clarify the mechanisms responsible for ultrastructural and histopathological changes occurring in the lungs of calves pre-infected with BVDV and challenged later with BHV-1, as well as to analyze the role of MΦs in the appearance of the lesions. Thus, this work will contribute to gaining a better understanding about how this virus predisposes to secondary airborne infections.
Materials and methods
The experimental design and collection of samples have been described by Risalde et al. . Briefly, twenty-four Friesian calves (8–9 months old), BVDV and BHV-1 antigen and antibody (Ab) free, were housed in the Animal Experimental Center of Cordoba University (Spain). The animals had an adjustment period of one week before the study started and were separated into three groups based on the inoculation protocol: twelve calves were inoculated intranasally with 10 mL of a suspension of non-cytopathic BVDV-1 strain 7443 with a titration of 105 tissue culture infective dose 50% (TCID50)/mL (courtesy of the Institute für Virologie, TIHO, Hannover, Germany). Twelve days later, when the calves showed neither clinical signs nor BVDV viraemia, they were challenged with 2 mL of BHV-1.1 strain Iowa containing 107 TCID50/mL (courtesy of the Hipra Laboratories, Girona, Spain) (BVDV/BHV-1 group). Two calves of this group, BHV-1.1-free, were used as BVDV infection controls. At the same time, ten calves were inoculated only with BHV-1.1 (BHV-1 group). Two un-infected (UI) animals were used as negative controls and received intranasally 2 mL of tissue culture fluid free of viruses. The time point of BHV-1.1 inoculation was defined as day 0.
After virus inoculation, clinical examination was performed daily. In order to determine the number of platelets, EDTA blood obtained from coccygeal vein and nasal swab samples were collected at 0, 3, 6, 9, 12, 15, 18 and 21 hours post-inoculation, 1, 2, 4, 5, 7, 9, 12 and 14 days post-inoculation (dpi) with BHV-1.1. The infected calves were sedated with xylazine (Rompun® 2% solution; Bayer Healthcare, Kiel, Germany) and euthanized by overdosing with thiopental-sodium (Thiovet®; Vet Limited, Leyland, Lancashire, UK) in batches of two at 1, 2, 4, 7 and 14 dpi. Two animals of the BVDV/BHV-1 group were killed at 0 dpi just before BHV-1.1 inoculation (BHV-1.1-UI), being used as BVDV infection controls for this group. On the contrary, the two negative control animals were euthanized at the end of the study, being used as UI controls for the BHV-1 group. The experimental procedure was carried out in accordance with the Code of Practice for Housing and Care of Animals used in Scientific Procedures, approved by the European Economic Community in 1986 (86/609/EEC amended by the directive 2003/65/EC).
All euthanized calves were subjected to necropsy examination. The samples were collected from the lungs and immediately frozen at −80°C for virological study; likewise, they were fixed in 10% buffered formalin solution for histopathological and immunohistochemical studies as well as in 2.5% glutaraldehyde in 0.1 M PBS for ultrastructural analysis.
Lung tissue samples were analyzed to discard any infection with secondary bacterial pathogens, being subjected to microbiological routine cultures (Xylose lysine deoxycholate agar, MacConkey agar and Blood agar) by using standard procedures. No bacteria were isolated from these samples.
Polymerase chain reaction (PCR)
RNeasy Lipid tissue kit (Qiagen) was used to extract BVDV ribonucleic acid (RNA) from lung samples, according to the manufacturer’s instructions. This RNA was subjected to a one step Real-Time Reverse Transcription plus Polymerase Chain Reaction (RT-PCR) using primers and Taqman probes (at the same concentration) from conserved regions of the 5’-UTR of BVDV-1a . The reaction was performed using the Real Time Ready RNA Virus Master (Roche, Mannheim, Germany) in a LightCycler 1.5, according to the manufacturer’s instructions. The samples with a cycle threshold value less than or equal to 45 were considered as positive. The positive control was the NCP BVDV-1 strain 7443 at 105 TCID50/mL.
A real-time PCR analysis of the BHV-1 deoxyribonucleic acid (DNA), extracted from lung samples using Genomic DNA from a tissue kit (Macherey-Nagel, Germany), was performed according to the OIE Terrestrial Manual . After analyzing the results on an Applied 7300 detector (Applied Biosystems, USA), any sample with a cycle threshold value less than or equal to 45 was considered as positive. The positive control was the BHV-1.1 strain Iowa at 108.3 TCID50/mL.
Tissue sample collection and processing for transmission electron microscopy (TEM)
Glutaraldehyde-fixed samples were post-fixed in 2% osmium tetroxide, dehydrated in acetone and embedded in Epon 812® (Fluka Chemie AG, Buchs, Switzerland). Sections (50 nm) were counterstained with uranyl acetate and lead citrate, and examined with a Philips CM-10 transmission electron microscope.
Formalin-fixed samples were dehydrated through a graded series of alcohol to xylol and embedded in paraffin wax by routine techniques for light microscopy. Samples were sectioned (3 μm) and stained by different methods as haematoxylin-eosin and the Fraser Lendrum technique, or processed for their immunohistochemical study using the avidin-biotin-peroxidase complex (ABC) method.
Detailed list of primary antibodies used in the immunohistochemical study
Antigen or cell detected
Monocytes and MΦs
Anti-murine iNOS/NOS Type II
iNOS/NOS Type II
BD Transduction Lab.
Anti-Human Von Willebrand Factor
Von Willebrand Factor
Tissue samples from cattle, in which reactivity for primary Ab against cytokines and cellular markers used in this study had been demonstrated, were used as positive controls in IHC [29, 38]. Tissue sections for which the specific primary Ab were replaced by rabbit or mouse non-immune sera (DakoCytomation, Glostrup, Denmark) were used as negative controls.
Cell counting and statistical analysis
In order to evaluate the number of immunolabeled cells and to correlate the results obtained using different Ab, two paraffin-wax blocks from the lungs of each animal were selected. Cell counts were carried out on 50 randomly chosen fields of 0.2 mm2 from tissue sections from these blocks. The results are given as the number of positive cells per 0.2 mm2. Identification of different types of immunolabeled cells was based on morphological features, location and size of the cells.
The different pulmonary MΦs exhibited rounded or elongated morphology, an indented nucleus and abundant cytoplasm. Interstitial MΦs (IMΦs), pulmonary intravascular MΦs (PIM) and pulmonary alveolar MΦs (PAM) are localized in distinct anatomical compartments of the lung, including connective tissue, being adhered to the endothelium in the pulmonary capillaries and air spaces, respectively. PIM and IMΦs were grouped together and are described as “septal MΦs”.
A semiquantitative estimation of platelet aggregation Factor-VIII-positive was performed on 50 randomly chosen 0.2 mm2 areas. The results are given as the presence of positive clusters of platelets per area as follows: absent (−), mild (+), moderate (++) and abundant (+++). For graphical representation and statistical analysis, they were scored from absent to severe (0 to 3).
Quantifications were performed by two experienced observers but with no previous knowledge of which group was being analyzed. Data were assessed to calculate mean ± standard error values and were analyzed with the SAS System for Windows, version 9.1 (SAS Institute, Cary, North Carolina, USA). Chi-square analysis was employed for the estimation of platelet aggregation Factor-VIII-positive for which a p < 0.05 was accepted as statistically significant, using a Fisher’s exact test in the instance of an expected value below 5. The Duncan’s Multiple Range Test (p < 0.05) was used to analyze significant differences of the values in the same group at various time points (*) and non-paired Student’s t-test (p < 0.05) was used between both inoculated groups at the same time point (**).
Respiratory signs and hematological findings
The main respiratory signs are given in Risalde et al. . Briefly, the calves pre-infected with BVDV show more intense respiratory signs such as cough, mucopurulent nasal discharge, dyspnoea and open-mouth breathing mainly between 4 and 11 dpi, while the calves of the BHV-1 group only presented a moderate serous nasal discharge.
Platelet numbers were within the clinically normal range in all calves throughout the study, except for 2 animals from both inoculated groups, where an important decrease of platelet numbers to approximately 57% was observed at 12 hpi.
Detection of BVDV and BHV-1 in the lungs
The presence of BVDV in the lungs of co-infected calves was detected by RT-PCR between 0 and 7 dpi (12–19 dpi BVDV), presenting the samples cycle threshold values between 33 and 39; whereas BHV-1 was detected by PCR from 2 to 14 dpi in these calves with cycle threshold values from 35 to 40. On the contrary, BHV-1 was only detected between 4 and 7 dpi in the BHV-1 group, showing the samples higher cycle threshold values (between 41 and 42), while BVDV was not detected in these calves throughout the study.
There were no remarkable lesions in the negative control animals. The pulmonary parenchyma of inoculated calves was affected by interstitial pneumonia with alveolar septal thickening produced by interstitial aggregates of mononuclear cells. The appearance of this alteration was earlier in the co-infected calves and was associated with occasional haemorrhages.
Subcellular changes indicative of a slight secretory activation in IMΦs such as enlargement, proliferation and dilation of rough endoplasmic reticulum and Golgi complex cisternae were mainly observed in the BHV-1 group at 4 dpi. Moreover, from 4 dpi, some PIM and PAM of both inoculated groups appeared enlarged and rounded, with loss of filopodia, increased number of lysosomes and varying amounts of cell debris in their cytoplasm, characteristic signs of phagocytic activation (Figure 1C).
Secretory activity of MΦs observed ultrastructurally was confirmed by IHC, which enabled the detection of MΦs-secreted inflammatory mediators.
Intracellular localization of COX-2 was perinuclear and cytoplasmic in the MΦs, being its expression scarce in the lungs during the study (Figure 8C), except for septal MΦs of the BVDV/BHV-1 group at 4 dpi (p < 0.0001) (Figure 7). This intense expression occurred at sites of inflammation and injury, mainly oedemas, and was correlated with the degree of pulmonary inflammation (Figure 8D).
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.
Avidin-biotin-peroxidase complex method
- BHV-1 group:
Calves inoculated only with BHV-1
Bovine respiratory disease complex
Bovine viral diarrhea virus
- BVDV/BHV-1 group:
Calves inoculated with BVDV and BHV-1
Inducible nitric oxide synthase
Nitric oxide synthase
Pulmonary alveolar MΦs
Polymerase chain reaction
Pulmonary intravascular MΦs
Reverse transcription-polymerase chain reaction
Tris buffered saline
Tissue culture infective dose 50%
Transmission electron microscopy
Tumor necrosis factor-α
BHV-1.1 un-infected animals.
This work was supported by grants from the Junta de Andalucía-FEDER (P09-AGR-4671). MA Risalde and V Molina were supported by a pre-doctoral grant from the Spanish Ministry of Education and Science. The authors thank the Institute für Virologie, TIHO (Hannover, Germany) and Laboratorios HIPRA SA (Girona, Spain) for providing the BVDV and BHV-1.1, respectively, and the farm “Las Rozuelas del Valle” (Torrecampo, Spain) for providing the animals. We specially thank to A. Muñoz for his technical assistance with the statistical analysis.
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