We thank Dr. Buczinski for his comments and compliments on our recent article, “Cardiac Troponin I in Calves with Congenital Heart Disease”. We understand and agree with his remarks, and wish to respond. Dr. Buczinski pointed that the cardiac troponin I (cTnI) concentration in plasma can be increased in cattle by various thoracic diseases or endotoxemia.
Therefore, we have investigated cTnI of calves with severe respiratory failure. Forty-one Holstein calves (mean±SD, 85.3 ± 46.1 days old; 10 females and 31 males) were enrolled in this study. Twenty healthy controls were deemed healthy on the basis of physical, biochemical, thoracic ultrasound, and radiological examinations. Twenty-one calves with severe clinical signs, including depression, coughing, nasal discharge, fever, and pulmonary wheezing sounds, were examined at Rakuno Gakuen University Veterinary Teaching Hospital and underwent bronchoscopy for diagnostic purposes. All calves with respiratory disease were euthanized because of poor prognosis within 1 week of hospitalization. The PCR for Mollicutes using bronchoalveolar lavage fluid detected Mycoplasma bovis in 1 sample (5%) from an apparently healthy calf and in 21 samples (100%) from calves with bronchopneumonia. Therefore, the statistical analysis focused on 19 healthy controls in which Mycoplasma bovis had not been detected and 21 calves with bronchopneumonia. A volume of 4 mL of whole blood was collected via jugular venipuncture into heparinized tubes for endotoxin and cTnI analyses and centrifuged at room temperature within 15 minutes of collection. The obtained plasma was separated and stored at -80°C until assay. To detect plasma endotoxin activity, kinetic turbidimetric assay (KTA)1 was performed on a 96-well microplate2 by using a microplate reader3 and endotoxin-measuring software.4 A standard curve was established for each assay in the range between 0.003 EU/mL and 3.0 EU/mL, according to the package insert of the LAL product. In this study, 20 fold diluted plasma samples were used for measuring endotoxin activity. Therefore, the lower limit of detection for this assay is 0.06 EU/mL. Plasma cTnI concentration was measured using a human cTnI 1-step sandwich EIA Kit (3rd generation),5 and conducted in accordance with the manufacturer's instructions. The lower limit of detection for this assay is 0.01 ng/mL. Plasma endotoxin activities and cTnI concentrations below 0.06 EU/mL and 0.010 ng/mL were statistically analyzed as 0.06 EU/mL and 0.010 ng/mL, respectively.
In healthy calves, plasma endotoxin activity was below the limit of detection in 16/19 (84%), with a median of 0.06 EU/mL (range, 0.060–0.231). The median plasma endotoxin activity (0.270 EU/mL; range, 0.098–8.068) was significantly higher in calves with bronchopneumonia than in controls by Mann-Whitney U-test (p < 0.001). Median cTnI concentrations in plasma in healthy calves and those with bronchopneumonia were below the limit of detection in 13/19 (68%) and 14/21 (67%), and were 0.010 ng/mL (range, 0.010–0.040) and 0.015 ng/mL (range, 0.010–0.030), respectively. From these data, plasma cTnI concentration did not differ between calves with and without bronchopneumonia, whereas calves with bronchopneumonia had significantly increased endotoxin activity compared with healthy animals. In addition, there was no relationship between endotoxin activity and cTnI concentration in plasma. We propose that the use of a cut-off value of ≥0.035 ng/mL cTnI in plasma will distinguish calves with and without significant congenital heart disease (CHD). In the calves with bronchopneumonia and high endotoxin activity, no case exceeded this cut-off value. It seems that the replacement of a control group of healthy calves by those with bronchopneumonia was better to establish the diagnostic accuracy in terms of specificity. ROC analysis was performed using these calves with bronchopneumonia as a control group. The proposed optimal cut-off point for cTnI with regard to Se and Sp (J) was determined to be 0.035 ng/mL (AUC = 0.846, p < 0.01, Se 71.4%, and Sp 94.7%). Therefore, using this proposed cut-off threshold (cTnI ≥0.035 ng/mL) to diagnose CHD in calves does not lead to over-diagnosing CHD in calves with simple infectious respiratory conditions or endotoxemia.
Dr. Buczinski has also pointed out that the small number of cases is a major problem in this paper. In addition, he pointed out from reference that there are differences in the normal range of plasma cTnI since different antibodies and equipment were used. We cannot provide a clear answer for these indications owing to the small number of cases. As such, further studies on the precision and accuracy are required.
In conclusion, our results suggest that using a cut-off value of ≥0.035 ng/mL cTnI in plasma may distinguish calves with and without significant CHD with reasonable Se and Sp.