Genomic and Proteomic Analysis of Stress and Disease Susceptibility
A common epidemiological feature of respiratory disease in fall-placed calves is that 15-30% of calves are diagnosed with acute infections and as many as 1-2% of calves develop fatal or chronic pneumonia. Furthermore, the majority of these respiratory infections cases develop within the first 30 days after calves enter the feedlot. Stress associated with weaning, transportation, social mixing, and dietary change has been identified as a factor that contributes significantly to respiratory disease susceptibility. It has been very difficult, however, to measure the impact of stress on either the severity or frequency of respiratory infections in beef cattle. For the present project we hypothesized that if an appropriate animal model was developed to investigate the effect of stress on bovine respiratory disease then it would be possible to identify specific gene and protein expression patterns characteristic of the stress response. Furthermore, specific changes in gene expression and protein profiles might be used to predict the outcome of bovine respiratory infections.
To address our hypothesis we used an experimental model of bovine respiratory disease to quantify the impact of stress on disease susceptibility in beef calves. For this model we selected cross-bred, ranch-reared calves which had no prior exposure to either bovine herpesvirus-1 (BHV-1) or Mannheimia haemolytica. BHV-1 is a major cause of viral respiratory infections in the feedlot and Mannheimia haemolytica is the major cause of fatal secondary bacterial respiratory infections or pneumonia in feedlot calves. The calves were first infected by inhaling an aerosol of BHV-1 and four days later the calves were exposed to an aerosol of Mannheimia haemolytica. Previous experiments at VIDO had shown that this combined viral-bacterial infection caused a fatal pneumonia in 40-50% of the calves. These experiments were repeated with 5 month old calves that were treated in two different ways to generate different types of stressors. One group of calves was weaned and adapted to a ration of hay and oats for two weeks prior to respiratory challenge. This group was referred to as the pre-conditioned (PC) group and these calves were exposed to the stress of transportation (3.5 hours) the day prior to BHV-1 challenge. The second group of age-matched, sucking calves were removed from their mothers and transported (3.5 hours) the day prior to BHV-1 challenge. This group was referred to as abrupt-weaned (AW) and represented a common management procedure within the beef industry where calves are weaned in the autumn, transported to auction markets, and then transported to a feedlot. A major observation in the present study was that the combined stressors of abrupt weaning and transportation resulted in a 100% increase or doubling of fatal respiratory infections. Transportation has been identified as a significant cause of stress in cattle but we observed that transportation alone did not significantly increase the expected rate of fatal respiratory disease in pre-conditioned calves. Therefore, we concluded that the combined stressors of weaning and transportation significantly increased the risk of fatal respiratory disease in beef calves.
Genomic and proteomic analyses were then used to determine if specific stress responses could be identified and if these responses correlated with either death or survival following respiratory infection with BHV-1 and Mannheimia haemolytica. There were no significant differences in the clinical responses of abrupt weaned and pre-conditioned calves and the level of BHV-I infection was not significantly altered. We observed, however, that the antiviral response was significantly increased in the abrupt weaned calves and that the magnitude of the antiviral response correlated directly with mortality following Mannhaeimia haemolytica infection. Our analysis of gene expression confirmed that calves with an enhanced antiviral response also had significantly elevated expression of receptors, such as toll-like receptor 4 (TLR4) and CD14, which play a key role in recognizing the presence of a bacterial infection. The relevance of this increased gene expression was confirmed when immune cells (monocytes) were isolated from calves following BHV-1 infection and Mannheimia haemolytica challenge. Stimulation of monocytes with lipopolysaccharide (LPS), a major cell wall component of Mannheimia haemolytica, confirmed that the capacity to produce pro-inflammatory cytokines, such as tumour necrosis factor (TNF) in response to LPS was directly correlated with disease outcome. These observations led to the conclusion that BHV-1 infection activates the innate immune system and this activation results in the profound immune pathology in the lung and possibly septic shock which causes fatal respiratory disease. These observations have significant implications for the treatment of acute bovine respiratory disease and indicate that antibiotic therapy may need to be combined with inhibitors of the innate immune system.
Genomic analysis of gene expression was complemented by proteomic, metabolomic, and elemental analyses of serum samples collected from pre-condition and abrupt weaned calves. Serum samples were collected immediately prior to BHV-1 challenge (Day 0) and immediately prior to Mannheimia haemolytica challenge on the fourth day after BHV-1 challenge (Day 4). The Day 0 time point was selected since serum analysis at this time was used to determine if there were significant physiological differences between AW and PC calves. The Day 4 time point was selected since serum analysis at this time addressed the question whether stress significantly altered the response of calves to viral infection. Proteomic and elemental analyses confirmed that there were significant physiological differences between AW and PC calves prior to BHV-1 infection. Furthermore, proteomic and elemental analysis confirmed that AW and PC calves displayed distinct responses to viral infection. In contrast, metabolomic analysis was unable to distinguish between the two treatment groups either before or after viral infection. Thus, proteomic and elemental analyses provided independent confirmation of the previous conclusion that the combined stressors of abrupt weaning and transportation significantly altered the response of calves to BHV-1 infection. Serum proteins associated with inflammation (haptoglobin; apolipoprotein) and serum iron were elevated in the abrupt weaned calves which is consistent with an enhanced capacity to respond to a secondary bacterial infection.
In conclusion, the present studies provided the first evidence that management related stressors significantly increase susceptibility to fatal respiratory disease in beef calves. Future studies will be required to determine if abrupt weaning alone is sufficient to enhance disease susceptibility or if a combination of stressors, such as transportation, social mixing, restraint, and weaning are required to enhance disease susceptibility. It may then be possible to design protocols to diminish the impact of individual stressors or avoid the combination of procedures which significantly enhance disease susceptibility.
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