The goal of this study was to identify a biomarker that predicts susceptibility to bovine respiratory disease in recently weaned and transported calves, at the time they arrive at the feedlot. Since the ideal biomarker would also play a mechanistic role in conferring susceptibility or resistance to disease, a second goal was to identify novel proteins in the epithelial lining fluid of the lower respiratory tract that determine susceptibility to pneumonia. We identified several proteins that were present at higher levels in the calves that remained healthy, compared to the calves that later developed pneumonia, and most of these proteins are known to function in resolution of inflammation. Among these proteins, annexin A1 and A2 showed a strong association with disease outcome, are known to be regulated by stress and glucocorticoids, may have a unique role in the pathogenesis of bovine respiratory disease, and are potential biomarkers for susceptibility to this disease in recently arrived, stressed feedlot cattle.
The study population was representative of feedlot production practices in much of North America, as the calves had been abruptly weaned in October and November, and transported to a different facility from five different sources. However, it should be acknowledged that the transportation time was 6 h or less, calves were not purchased through an auction, and mixing of calves from different sources was limited. Antimicrobial treatment was not given until clinical signs developed, so the findings are most relevant to disease control strategies that avoid metaphylactic use of antimicrobial drugs.
Calves were considered to be clinically healthy at the time of sampling, 2–3 days after arrival. Although nasal discharge and hyperemia of the nasal and tracheal mucosa was observed in a few animals, clinical signs of pneumonia were not detected at this on-arrival time point, and none of these calves had elevations in rectal temperature, serum haptoglobin concentrations, or blood neutrophil counts.
Clinical diagnosis of pneumonia is problematic in cattle . In this study, the positive and negative selection criteria were based on evaluation of clinical signs by the experienced feedlot operator, confirmation of the clinical diagnosis by a veterinarian, elevated rectal temperature, and increased serum haptoglobin levels. Calves were only included in the study groups “Later developed pneumonia” or “Calves that remained healthy” if all of these factors were present or absent, respectively, to increase confidence in the diagnosis. Other calves had conflicting clinical and laboratory data, and were not included in the DIGE analysis.
Although we recognize that the difficulty in acquiring BALF makes it unsuitable for routine screening of feedlot cattle, our analysis was based on BALF because it is the more appropriate sample for detecting changes in the epithelial lining fluid of the distal respiratory tract [4, 8, 9]. Initial work was done with 2-dimensional electrophoresis (data not shown), but DIGE was used in the present study because of superior sensitivity and reproducibility for the identification of proteins in the BALF.
Among those differentially expressed proteins, annexin A1 and A2 were of particular interest. Other differentially expressed proteins were macrophage capping protein, calcyphosin, dihydrodiol dehydrogenase 3, peroxiredoxin I, and superoxide dismutase. Three of these are antioxidants: dihydrodiol dehydrogenase 3 is an aldoketoreductase that maintains cellular redox homeostasis, peroxiredoxin I catalyzes hydrogen peroxide and also has anti-inflammatory functions, and superoxide dismutase scavenges superoxide anion and attenuates lung inflammatory responses . Since the lung encounters considerable oxidative stress, animals having higher levels of these antioxidants and anti-inflammatory proteins may be protected from tissue damage caused by bacterial infection. Macrophage capping protein, also named Cap G and cofilin, is a calcium-sensitive protein expressed in alveolar macrophages [11, 12], and has roles in rearrangement of actin filaments, membrane ruffling, and complement- and immunoglobulin-mediated phagocytosis . Calcyphosin is a calcium-binding protein of uncertain function: it inhibits mast cell degranulation, has anti-allergic properties in bovine lung , and is involved in cAMP signaling , cell proliferation and differentiation .
The differential expression of annexins A1 and A2 were of particular interest, because of the number of differentially expressed spots corresponding to these proteins, the magnitude and consistency of the differences, and the apparent relevance to the pathogenesis of bovine respiratory disease. Annexins A1 and A2 were found at higher levels in the BALF of calves that remained healthy compared to those that later developed pneumonia, and these DIGE and mass spectrometry findings were confirmed by Western blot analysis of the same BALF samples. The Western blot analysis revealed 3 isoforms of annexin A1 and two isoforms of annexin A2. This preliminary analysis also revealed considerable variation between animals in annexin A1 and A2 isoforms, and additional investigations will be needed to determine the reasons for this variability and the relationship with disease susceptibility.
Annexins A1 and A2 are expressed in bronchial epithelial cells of cattle . Annexins are also found in extracellular tissue fluids, and annexin A2 is detected on the surface of endothelial cells [18, 19]. Glucocorticoids induce expression of both the annexin A1 gene and the annexin receptor ALXR [20, 21], and effect rapid translocation of annexin A1 to the cell surface . Similarly, annexin levels are greater in hyperadrenocorticism and in response to stress [23, 24]. Annexin A1 mediates some of the anti-inflammatory effects of glucocorticoids: it binds to the surface of neutrophils, inducing loss of L-selectin and neutrophil apoptosis , and acts on macrophages to promote phagocytosis and removal of apoptotic cells . These functions of annexin A1 reduce neutrophil extravasation and accumulation in the tissue .
Of relevance to feedlot cattle, we previously found that annexin A1 increased significantly in the BALF of beef calves after transportation stress , and a preliminary analysis in the current study confirmed a positive correlation between serum cortisol concentration and annexin A1 levels in BALF. Thus, it is plausible that annexin A1 may be a biomarker of stress-associated susceptibility to bovine respiratory disease. However, we found increased levels of annexin A1 in calves that remained healthy compared to those that later developed disease, yet glucocorticoids are expected to induce annexin expression. Therefore, stress does not simply result in both annexin A1 induction and increased susceptibility to pneumonia; there is likely a more complex relationship between stressful experiences, synthesis of glucocorticoids, levels of annexin A1 in the epithelial lining fluid of the airways, and susceptibility to pneumonia.
Annexin A2 plays a vital role in several biological processes, including matrix degradation , preventing angiogenesis , cellular protection against oxidative injury , repair of airway epithelial cells , and the pro-inflammatory effects of tissue plasminogen activator on macrophages . However, the most recognized role of annexin A2 is in promoting fibrinolysis. Annexin A2 is a receptor for plasminogen and tissue plasminogen activator on the surface of endothelial cells, resulting in activation of plasmin and initiation of fibrinolysis .
These findings of higher levels of annexins A1 and A2 in calves that remained healthy after arrival to the feedlot suggest a role of annexins in the pathogenesis of bovine respiratory disease. During M. haemolytica pneumonia, there is a strong influx of neutrophils and macrophages, and accumulation of fibrin in the lung. These cascades of events lead to neutrophil-induced tissue injury, and impair alveolar ventilation and gas exchange . The data suggest that calves with high levels of annexin A1 may be better able to control neutrophil infiltration and promote neutrophil apoptosis in the lung, which could prevent neutrophil-induced lung injury and dampen the systemic consequences of endotoxemia. Similarly, the findings suggest that calves having higher levels of annexin A2 may be better able to degrade the fibrin that forms within the pulmonary alveoli, which may reduce the severity or progression of disease following M. haemolytica infection.
The present study was not designed to identify the mechanism for differing annexin levels in susceptible vs. resistant calves. Additional work will be needed to evaluate the cellular source of annexins in the lung, the effect of genetics and individual differences on baseline levels of annexin proteins, and the impact of infection and weaning/transportation stress on annexin levels. Thus, characterizing what role these differing annexin levels have in the pathogenesis of bacterial pneumonia will require additional investigation. Nonetheless, annexins A1 and A2 show promise as novel biomarkers of disease susceptibility in recently arrived feedlot cattle. Such biomarkers could be useful for quantification of stress responses under varying management conditions, and accurate classification of recently arrived feedlot cattle with respect to risk of bovine respiratory disease, to facilitate group-specific disease control strategies.