Despite progress in veterinary medicine, total or partial failure of passive transfer (FPT) still commonly occurs with prevalence ranging between 11 and 31% in North America in beef calves, with prevalence of approximately 40% in dairy calves. Calves presenting with FPT are more susceptible to infectious diseases and have higher morbidity and mortality rates. The detection of FPT, at an individual level as well as at herd level, therefore is fundamental for improving the health and management of calves. The use of refractometry for detecting FPT frequently was investigated because this method is inexpensive, quick, and easy to perform under farm conditions. The refractometric measurement of serum total protein concentration is based on the refraction index (n) of the serum and its conversion to serum total protein concentration using a known conversion factor. Depending on the manufacturer, 2 different conversion factors are applied, 1 determined by Wolf and the other by the Atago Corporation. The Wolf conversion factor is reputed to give higher serum total protein concentrations compared with the Atago conversion factor. As the index of refraction is influenced by the temperature of the solute, Automatic Temperature Compensation (ATC) refractometers were commercialized to avoid the impact of potential temperature variations on the results. Recently, digital refractometers have been available on the market but, to our knowledge, only 1 study has been performed to assess FPT with this type of refractometer.
The objectives of this study were to compare the accuracy of 4 different refractometers for measuring serum total protein concentration in beef calves and, based on the serum IgG concentration threshold of 1,600 mg/dL, to determine the optimal threshold of serum protein concentration above which adequate passive transfer can be concluded. In addition, the gamma-glutamyl transferase (γ-GT) activity and the total immunoglobulin (Ig) concentration in serum were compared with the serum IgG concentration for assessment of passive transfer in beef calves.
- Top of page
- Materials and Methods
All refractometers had the same accuracy for serum total protein concentration assessment in calves despite a significantly higher bias for the Wolf ATC. Choosing the serum IgG of 1,600 mg/dL as threshold for diagnosing FPT, the use of the specific serum protein threshold for each refractometer allows the investigation of FPT by the bovine practitioner. Ig is highly correlated with the IgG concentration, whereas γ-GT activity is poorly correlated with the IgG concentration.
The high correlation coefficient between the refractometric and biuret measurements (0.953–0.964) obtained with each refractometer indicates a nearly linear relationship. To our knowledge, few data concerning the comparison between refractometric and biuret methods in cattle are available in the literature. Quigley described a correlation coefficient of 0.92 for calves aged about 3–5 days, but all measurements were performed on plasma, and not on serum. In adult cattle, McSherry and Al-Baker reported a correlation coefficient of 0.982, and Caprita and Caprita reported a correlation coefficient of 0.990 for plasmatic protein values, which are similar to results obtained in the present study.
In our study, correlation coefficients were not significantly different among the 4 refractometers, indicating similar accuracy for measuring serum total protein concentrations. This result is in agreement with the study of Calloway et al, which identified a similar ability to detect FPT with 3 different refractometers, and with the study of Wallace et al, which found a correlation coefficient of 0.980 between the serum total solid concentrations measured by 2 different refractometers (Atago ATC versus digital refractometer). In contrast to the findings of George, no significant difference of serum total protein concentration was found between the Wolf and the Atago refractometers. This absence of significant difference could be explained by the range of measurements performed in our study. Indeed, according to George, Atago refractometers give approximately 5 g/L lower protein results, but this occurs mainly in the 5–30 g/L range of measurement.
Our results show that compared with the biuret method, all refractometers underestimated the serum total protein concentrations by about 2.7–5.0 g/L. This difference in protein measurement agrees with the study of Green et al, in which mean total protein values measured in cattle by the biuret method were higher by about 3 g/L compared with results obtained by refractometry. This bias could be explained by the heterogeneity of the serum proteins involved, leading to interference with the specific technique of measurement of both methods.
The bias obtained with the Wolf ATC refractometer was significantly higher than that obtained using the other refractometers. This finding is probably because of lower protein results obtained with the Wolf ATC refractometer compared with the other refractometers, although the refractometric measurements between the Wolf ATC and the other refractometers were not significantly different. Moreover, the difficulty of correctly visualizing the demarcation line with the Wolf ATC refractometer during measurement could increase the risk of erroneous results. For all combinations, median sensitivities and specificities obtained with the Bayesian model of test accuracy were relatively close, indicating that these 2 methods of protein measurements were conditionally dependent. This conditional dependence reflects the high correlation coefficients obtained between the biuret method and refractometry using the Bland–Altman method. Even if values of sensitivities and specificities obtained with noninformative prior data are elevated, they could be improved by the use of more precise prior data.
The high values of intraclass correlation coefficient (ICC) indicate excellent reliability of the serum protein measurement with each refractometer, under farm conditions. In our study, the serum IgG concentration of 1,600 mg/dL was chosen as the threshold point below which calves were considered to have FPT. This IgG concentration is much higher than the generally used concentration of 1,000 mg/dL. However, the choice of an appropriate serum IgG threshold concentration for the maintenance of health in calves depends on many factors, such as the environment, the presence of infection, the breed or the prevalence of FPT in the studied population. For example, several researchers have suggested serum IgG concentrations of 500 mg/dL and 800 mg/dL for decreasing mortality linked to septicemia. Likewise, for decreasing preweaning morbidity and mortality, serum IgG concentrations of 800 mg/dL, 1,000 mg/dL, 1,600 mg/dL, and even 2,400 mg/dL are found in the literature.
Our choice of a higher serum IgG concentration was motivated by the good colostral immunity transfer observed in these Belgian Blue calves resulting in a low number of calves (n = 2) having serum IgG concentration <1,000 mg/dL. Serum total protein concentration threshold values obtained in our study are higher than the value of 52 g/L generally used, but this concentration was determined for a serum IgG concentration of 1,000 mg/dL and mainly in dairy calves where FPT occurs more frequently.
The sensitivity, the specificity, the AUC, proportion of correctly classified calves, and Kappa statistic were not significantly different among refractometers using their individual serum concentration protein thresholds. However, under field conditions, it is easier to use only 1 serum protein concentration threshold, independently of the type of refractometer used. When using the same serum protein concentration threshold of 56 g/L for the 4 refractometers, sensitivity and specificity will only change for the Atago ATC (Se = 88.8%, Sp = 93.3%) and for the Wolf ATC refractometers (Se = 100%, Sp = 70%). When this serum protein concentration threshold is used for the 4 refractometers, the digital ATC (followed by the Atago) is the most accurate with the best sensitivity and specificity.
Specificities obtained with our serum protein concentration thresholds are in the same range as those obtained by Calloway et al, whereas sensitivities are higher. Nevertheless, it is possible that with temperature variations at the time of measurement, sensitivity and specificity of the non-ATC Atago refractometer would change, with probably a decrease in its accuracy.
The AUC between 0.984 and 0.989 indicates very good performance of refractometry for diagnosing FPT in calves. The low prevalence of FPT observed in our study (about 17%) leads to a high proportion of calves being correctly classified. The kappa statistic values between 0.75 and 0.82 indicate good agreement among the refractometers for the diagnosis of FPT in calves.
The low correlation coefficient between IgG and γ-GT activity indicates that γ-GT activity is not a valuable test for the evaluation of the passive transfer in calves. Perino et al and Wilson et al obtained similar results with correlation coefficients of 0.41 and 0.438, respectively. However, Parish et al and Güngör et al found better correlation coefficients of 0.63 and 0.57, respectively, for dairy calves <10 days of age. One explanation for this disparity of results could be the important interindividual variations of γ-GT activity in the dams’ colostrum and the absence of significant correlation of γ-GT activity between calf plasma and ingested colostrum. Perino et al suggest that the γ-GT activity does not indicate the amount of colostrum absorbed, and that some degree of activity >200 IU/L simply reflects that the calf has absorbed some colostrum.
As described in another study, the high correlation coefficient (0.956) between serum IgG and Ig obtained in our calves indicates a very good linear relationship. In fact, Ig represents the electrophoretic portion that contains the immunoglobulins transferred via the colostrum. To our knowledge, the fusion of the beta-2 and gammaglobulin peaks during the first days of life, and the separation of these 2 peaks during the first week were only described by Godeau et al. Therefore, the interpretation of the serum Ig obtained by electrophoresis for the assessment of passive transfer during the first week of life must take the age of the calves into account. Additional studies are needed for a better understanding of this peak fusion and separation during the first week of life.