Exhaled breath condensate for the diagnosis of bovine respiratory disease
Bovine respiratory disease (BRD) is a leading cause of economic loss to the beef and the dairy sectors in Ontario on an annual basis. This project adapted an existing medical technology (device for collection of exhaled breath condensate from people), for application in the beef sector to address the lack of a reliable method to accurately detect pneumonia in beef cattle. Exhaled breath condensate (EBC) is a readily obtained clinical sample that is derived from the fluid lining the respiratory tract. Exhaled breath is collected in a cooled unit to condense expired droplets and gases into liquid. The resulting fluid is then tested for markers of inflammation that would suggest the presence of lung disease. In this study, the commercially-available human collection device, RTube (Respiratory Research, Inc, Austin, TX) was successfully adapted to use in weanling cattle. The modified collection system was non-invasive, very user-friendly, portable, and could be disinfected between animals. The materials used to modify the collection device are commercially available and can be purchased at low cost. This technology could therefore be used on-farm by bovine veterinarians or producers and would have an applied and practical outcome, allowing for non-invasive sampling of the respiratory system for more timely and accurate diagnosis of BRD in the farm/feedlot setting.
In order to assess the utility of exhaled breath condensate to differentiate healthy cattle from cattle with pneumonia, we conducted an experimental disease study on weanling bull calves that were randomly allocated to either control (n=5) or infected (n=6) groups. Calves in the control group received sham challenge of saline instilled into the distal trachea, whereas calves in the infected group were challenged with the bacterium, Mannheimia haemolytica. Ultrasound examination of the chest was performed to track the onset of lung lesions post-challenge. Blood and exhaled breath condensate samples were obtained from all calves serially over time, until day 5 after challenge at which time all calves were euthanized and gross post mortem examination was done to verify and characterize any lung lesions.
The lung at intercostal spaces 1 – 10 of each lung field was evaluated using a variable frequency linear rectal transducer (6.2 MHz; depth 9 cm) with alcohol as the transducing agent. Moderate-to-severe lung consolidation was first noted 2 hours post-inoculation, time to severe consolidation (consolidated area > 3cm x 1cm) was 6 hours, and maximum lesions were noted 24 hours after challenge. The ultrasound results suggest that lung consolidation occurs soon after infection, progresses rapidly, and can be diagnosed ante-mortem by ultrasonography.
The acute phase proteins haptoglobin and fibrinogen were measured in blood from calves in both groups. Increases in fibrinogen and haptoglobin were observed only in the infected calves, with peak values seen at approximately 18 – 24 hours post-challenge. This was consistent with the time of maximal lung lesions as seen on ultrasound examination. There was a more robust change in blood haptoglobin seen between the sick and healthy calves (versus fibrinogen), therefore haptoglobin was selected as the acute phase protein to initially test in the exhaled breath condensate samples.
Haptoglobin concentration in EBC was assessed using existing laboratory methodology (photometric) at the Animal Health Laboratory, University of Guelph. No haptoglobin was detected in the samples, although there was concern that the test method was not sufficiently sensitive to detect the low concentrations of haptoglobin potentially present in EBC. Therefore, a second method to measure haptoglobin was investigated using a commercially-available ELISA kit (Immunoperoxidase ELISA for Bovine Haptoglobin, Immunology Consultants Laboratory Inc, Portland, OR). Several EBC samples from timepoints in the study that matched peak serum haptoglobin values were selected to test using this ELISA. Values in the EBC samples were similar to blank sample values, suggesting that no haptoglobin was present in the EBC. To ensure that the ELISA test kit could detect haptoglobin in the presence of EBC, baseline (pre-infection) EBC samples were spiked with varying concentrations of commercially-available haptoglobin and the ELISA was repeated. Haptoglobin was reliably detected in the spiked samples even at extremely low concentrations (lower limit of detection was determined to be 0.1 ng/ml). From this, it was concluded that lung inflammation from bacterial pneumonia does not result in detectable haptoglobin in exhaled breath of affected calves. Aliquots of EBC from an infected calf were also assessed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) with silver staining for protein detection. Since no protein was detected in the EBC using SDS-PAGE, efforts to test other acute phase proteins as inflammatory markers in EBC were not pursued.
The EBC samples have been frozen and stored for future investigation of their composition (eg. mass spectrometry) in order to potentially identify other inflammatory markers outside of protein fractions.
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